reca  (New England Biolabs)


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    Name:
    RecA
    Description:
    RecA 1 000 ug
    Catalog Number:
    M0249L
    Price:
    288
    Category:
    Other Endonucleases
    Size:
    1 000 ug
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    New England Biolabs reca
    RecA
    RecA 1 000 ug
    https://www.bioz.com/result/reca/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    reca - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "Residues in the fingers domain of the translesion DNA polymerase DinB enable its unique participation in error-prone double-strand break repair"

    Article Title: Residues in the fingers domain of the translesion DNA polymerase DinB enable its unique participation in error-prone double-strand break repair

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.006233

    Highly conserved fingers domain residue is important for DinB's activity during strand exchange. A, schematic shows experimental setup for RecA-dependent strand-exchange experiments. Fluorescently-labeled dsDNA ( left ) is mixed with each of a set of ssDNA RecA filaments ( right ). Each in the set of filaments contains a different length of homology ( N ) to the labeled dsDNA (signified by gray, green, magenta, and blue lines ). In the dsDNA, the template strand is labeled with rhodamine ( yellow circle ), and the displaced strand is labeled with fluorescein ( white star ). These fluorophores are located 5 bp away from area of homology between dsDNA and ssDNA RecA filament (Δ L = 5 bp). The proximity of the rhodamine quenches fluorescein fluorescence until template and displaced strands are separated. Fluorescence increases when the ssDNA RecA filament invades the dsDNA, and DinB synthesizes DNA using the ssDNA filament as a primer. Five nucleotide insertions are needed to separate the displaced strand at the location of the fluorescent labels and relieve quenching. B, when only RecA or only DinB is mixed with the highest homology ssDNA RecA filament ( N = 75, blue filament in A ), the fluorescent labels are not efficiently separated, indicating that the dsDNA is still annealed at the location of the labels. When both proteins are present in the absence of dCTP, dGTP, and dTTP (dATP is present for nucleoprotein filament assembly), baseline fluorescence is observed. A RecA filament with full homology to the dsDNA ( N = 90) is used to determine maximum possible fluorescence in the assay. C, DinB stabilizes strand exchange in a homology-dependent manner. As homology increases between the ssDNA RecA filament and the fluorescently-labeled dsDNA, DinB efficiently separates the dsDNA at the location of the fluorescent labels. This indicates that increased homology allows DinB to more efficiently stabilize strand-exchange products. D, DinB(C66A) stabilizes strand-exchange products, but it does so with less efficiency than the native enzyme. N indicates the length of homology between dsDNA and ssDNA filament; Δ L indicates the distance between region of homology on dsDNA and fluorescent label; D init indicates the distance between fluorescent label and closet end of dsDNA; Δ F indicates the change in fluorescence measured in counts/s with respect to the fluorescence at 0 s. Experiments were performed in triplicate with similar results. Representative data are shown.
    Figure Legend Snippet: Highly conserved fingers domain residue is important for DinB's activity during strand exchange. A, schematic shows experimental setup for RecA-dependent strand-exchange experiments. Fluorescently-labeled dsDNA ( left ) is mixed with each of a set of ssDNA RecA filaments ( right ). Each in the set of filaments contains a different length of homology ( N ) to the labeled dsDNA (signified by gray, green, magenta, and blue lines ). In the dsDNA, the template strand is labeled with rhodamine ( yellow circle ), and the displaced strand is labeled with fluorescein ( white star ). These fluorophores are located 5 bp away from area of homology between dsDNA and ssDNA RecA filament (Δ L = 5 bp). The proximity of the rhodamine quenches fluorescein fluorescence until template and displaced strands are separated. Fluorescence increases when the ssDNA RecA filament invades the dsDNA, and DinB synthesizes DNA using the ssDNA filament as a primer. Five nucleotide insertions are needed to separate the displaced strand at the location of the fluorescent labels and relieve quenching. B, when only RecA or only DinB is mixed with the highest homology ssDNA RecA filament ( N = 75, blue filament in A ), the fluorescent labels are not efficiently separated, indicating that the dsDNA is still annealed at the location of the labels. When both proteins are present in the absence of dCTP, dGTP, and dTTP (dATP is present for nucleoprotein filament assembly), baseline fluorescence is observed. A RecA filament with full homology to the dsDNA ( N = 90) is used to determine maximum possible fluorescence in the assay. C, DinB stabilizes strand exchange in a homology-dependent manner. As homology increases between the ssDNA RecA filament and the fluorescently-labeled dsDNA, DinB efficiently separates the dsDNA at the location of the fluorescent labels. This indicates that increased homology allows DinB to more efficiently stabilize strand-exchange products. D, DinB(C66A) stabilizes strand-exchange products, but it does so with less efficiency than the native enzyme. N indicates the length of homology between dsDNA and ssDNA filament; Δ L indicates the distance between region of homology on dsDNA and fluorescent label; D init indicates the distance between fluorescent label and closet end of dsDNA; Δ F indicates the change in fluorescence measured in counts/s with respect to the fluorescence at 0 s. Experiments were performed in triplicate with similar results. Representative data are shown.

    Techniques Used: Activity Assay, Labeling, Fluorescence

    DinB(C66A) variant is deficient in RecA-independent strand displacement. A, graphic depiction of the DNA substrate used in these experiments. A 29-nt ssDNA primer ( gray line ) was annealed to a 90-nt template ( bottom black line ) as well as to a 75-nt fluorescently-labeled oligonucleotide ( top black line with the fluorophore represented by the star ). The 75-nt oligonucleotide displaced strand is composed of a 61-nt complementary to the 90-bp template and of a 14-nt unannealed flap located at the 5′ end. The fluorescein label (depicted by the star ) on the displaced strand was located on the first nucleotide of the 75-bp complementary region. If displaced and template strands are separated, fluorescence is altered. DinB must insert a single nucleotide onto the end of the primer to displace the labeled nucleotide. B, experiments with all dNTPs added show that DinB stabilizes strand displacement after a short lag (highlighted by the enlargement in C ) with greater efficiency than the DinB(C66A) variant. Initial velocities for both proteins from 0 to 50 s are not significantly different from zero. The rate of the DinB reaction from 100 to 200 s (46.40 ± 1.188 cps) is significantly higher than the velocity of the DinB(C66A) reaction at the same time point (11.80 ± 1.140 cps; p value
    Figure Legend Snippet: DinB(C66A) variant is deficient in RecA-independent strand displacement. A, graphic depiction of the DNA substrate used in these experiments. A 29-nt ssDNA primer ( gray line ) was annealed to a 90-nt template ( bottom black line ) as well as to a 75-nt fluorescently-labeled oligonucleotide ( top black line with the fluorophore represented by the star ). The 75-nt oligonucleotide displaced strand is composed of a 61-nt complementary to the 90-bp template and of a 14-nt unannealed flap located at the 5′ end. The fluorescein label (depicted by the star ) on the displaced strand was located on the first nucleotide of the 75-bp complementary region. If displaced and template strands are separated, fluorescence is altered. DinB must insert a single nucleotide onto the end of the primer to displace the labeled nucleotide. B, experiments with all dNTPs added show that DinB stabilizes strand displacement after a short lag (highlighted by the enlargement in C ) with greater efficiency than the DinB(C66A) variant. Initial velocities for both proteins from 0 to 50 s are not significantly different from zero. The rate of the DinB reaction from 100 to 200 s (46.40 ± 1.188 cps) is significantly higher than the velocity of the DinB(C66A) reaction at the same time point (11.80 ± 1.140 cps; p value

    Techniques Used: Variant Assay, Labeling, Fluorescence

    2) Product Images from "Opposing Roles for Two Molecular Forms of Replication Protein A in Rad51-Rad54-Mediated DNA Recombination in Plasmodium falciparum"

    Article Title: Opposing Roles for Two Molecular Forms of Replication Protein A in Rad51-Rad54-Mediated DNA Recombination in Plasmodium falciparum

    Journal: mBio

    doi: 10.1128/mBio.00252-13

    (A) Role of PfRPA1L and PfRPA1S during SSE activity of PfRad51. (I) PfRad51 and SSB. (II) PfRad51 and PfRPA1L. (III) PfRad51 and PfRPA1S. (B) PfRPA1S downregulates the function of PfRPA1L. (I) PfRad51, 0.5 μM PfRPA1L, and 0.5 μM PfRPA1S. (II) PfRad51, 0.5 μM PfRPA1L, and 0.75 μM PfRPA1S. (III) PfRad51, 0.5 μM PfRPA1L, and 1.0 μM PfRPA1S. (IV) PfRad51, 0.5 μM PfRPA1L, and 2.0 μM PfRPA1S. (V) PfRad51 and 0.5 μM PfRPA1S preincubated for 10 min, followed by addition of 0.5 μM PfRPA1L. (VI) PfRad51 and 0.5 μM PfRPA1S preincubated for 10 min, followed by addition of 1.0 μM of PfRPA1L. (C) Role of PfRad51, PfRPA1L, and PfRPA1S in the presence of the bacterial homologue RecA and SSB. (I) PfRad51, 0.5 μM SSB, and 0.5 μM PfRPA1S. (II) RecA and PfRPA1L. (III) RecA and PfRPA1S. (IV) RecA, 0.5 μM PfRPA1L, and 0.5 μM PfRPA1S. Aliquots were collected at time points (min) indicated above each lane and quenched with stop solution, and products were revealed on 1% TAE agarose gel, followed by EtBr staining. Lds, linear double-stranded DNA; NC, nicked circular dsDNA; JM, joint molecule. These figures are a representative assay of three biologically independent strand exchange assays.
    Figure Legend Snippet: (A) Role of PfRPA1L and PfRPA1S during SSE activity of PfRad51. (I) PfRad51 and SSB. (II) PfRad51 and PfRPA1L. (III) PfRad51 and PfRPA1S. (B) PfRPA1S downregulates the function of PfRPA1L. (I) PfRad51, 0.5 μM PfRPA1L, and 0.5 μM PfRPA1S. (II) PfRad51, 0.5 μM PfRPA1L, and 0.75 μM PfRPA1S. (III) PfRad51, 0.5 μM PfRPA1L, and 1.0 μM PfRPA1S. (IV) PfRad51, 0.5 μM PfRPA1L, and 2.0 μM PfRPA1S. (V) PfRad51 and 0.5 μM PfRPA1S preincubated for 10 min, followed by addition of 0.5 μM PfRPA1L. (VI) PfRad51 and 0.5 μM PfRPA1S preincubated for 10 min, followed by addition of 1.0 μM of PfRPA1L. (C) Role of PfRad51, PfRPA1L, and PfRPA1S in the presence of the bacterial homologue RecA and SSB. (I) PfRad51, 0.5 μM SSB, and 0.5 μM PfRPA1S. (II) RecA and PfRPA1L. (III) RecA and PfRPA1S. (IV) RecA, 0.5 μM PfRPA1L, and 0.5 μM PfRPA1S. Aliquots were collected at time points (min) indicated above each lane and quenched with stop solution, and products were revealed on 1% TAE agarose gel, followed by EtBr staining. Lds, linear double-stranded DNA; NC, nicked circular dsDNA; JM, joint molecule. These figures are a representative assay of three biologically independent strand exchange assays.

    Techniques Used: Activity Assay, Agarose Gel Electrophoresis, Staining

    3) Product Images from "The positioning of Chi sites allows the RecBCD pathway to suppress some genomic rearrangements"

    Article Title: The positioning of Chi sites allows the RecBCD pathway to suppress some genomic rearrangements

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1252

    Schematic of a RecBCD-dependent repair of a double-strand break and a genomic rearrangement that might result. ( A ) i . The dsDNA sequences are identical. The DSB is indicated by the gray arrow. ii . The bright green triangles indicate the Chi sites on each strand that are oriented so that they would be recognized by RecBCD as it proceeds along the dsDNA from the DSB. The L χleft and L χright are the separations between the position of the DSB and the nearest appropriately oriented Chi site on each initiating strand. iii . RecBCD creates the two initiating ssDNAs, while the complementary strands are degraded or looped (dotted circles). L left and L right are the distances separating the DSB from the 3′ end of each initiating strand. Note that L left ≥ L χleft and L right ≥ L χright . iv . RecA mediated strand exchange creates heteroduplex products that reach the 3′ ends of the filaments . v . DNA polymerase (orange rectangles) extends both initiating ssDNAs by copying the complementary strands beginning at the 3′ end of an initiating strand in a RecA filament. ( B ) The red and blue lines represent two identical dsDNAs that include two copies of a repeated sequence, Copy 1 and Copy 2 separated by a non-repeated region. i . A DSB occurs near the center of Copy 1. ii . After the DSB, RecBCD creates two ssDNA–RecA filaments by loading RecA onto the initiating ssDNA created by RecBCD. The two ssDNA–RecA filaments are indicated by the green horizontal arrows pointing toward the 3′ end. iii . Both filaments pair with the sequence matched regions in Copy 2 and form sequence matched heteroduplex products that extend to the 3′ ends of the initiating strands. DNA synthesis then completes two dsDNAs. The sequence region that includes the heteroduplex products and the newly synthesized DNA is shown in purple. The completion of the dsDNA is followed by Holliday junction resolution that is a crossover on the right side. As a result, the upper dsDNA lacks the sequence region between the two copies, and the lower dsDNA has two copies of the region between the two repeats. The duplicated regions are highlighted by the yellow rectangles. ( C ) Schematic of a DSB that occurs in Copy 1 of a repeated sequence. The appropriately oriented Chi sites are indicated by the bright green triangles, and the direction of RecBCD is shown by the black arrows. Copy 1 only includes appropriately oriented Chi sites in the dark red strand, so only the dark red strand can terminate in a region of Copy 1. ( D ) Same as C), but the DSB occurs to the left of Copy 1, which changes the number of correctly oriented Chi site in this region. The light pink filament cannot include a region of Copy 1 because RecBCD moves away from the DSB. ( E ) In a different sequence than C), Copy 1 includes appropriately oriented Chi sites on both strands, allowing the rearrangement shown in B); however, this never occurs in any of the 12 enteric bacteria we studied in this work.
    Figure Legend Snippet: Schematic of a RecBCD-dependent repair of a double-strand break and a genomic rearrangement that might result. ( A ) i . The dsDNA sequences are identical. The DSB is indicated by the gray arrow. ii . The bright green triangles indicate the Chi sites on each strand that are oriented so that they would be recognized by RecBCD as it proceeds along the dsDNA from the DSB. The L χleft and L χright are the separations between the position of the DSB and the nearest appropriately oriented Chi site on each initiating strand. iii . RecBCD creates the two initiating ssDNAs, while the complementary strands are degraded or looped (dotted circles). L left and L right are the distances separating the DSB from the 3′ end of each initiating strand. Note that L left ≥ L χleft and L right ≥ L χright . iv . RecA mediated strand exchange creates heteroduplex products that reach the 3′ ends of the filaments . v . DNA polymerase (orange rectangles) extends both initiating ssDNAs by copying the complementary strands beginning at the 3′ end of an initiating strand in a RecA filament. ( B ) The red and blue lines represent two identical dsDNAs that include two copies of a repeated sequence, Copy 1 and Copy 2 separated by a non-repeated region. i . A DSB occurs near the center of Copy 1. ii . After the DSB, RecBCD creates two ssDNA–RecA filaments by loading RecA onto the initiating ssDNA created by RecBCD. The two ssDNA–RecA filaments are indicated by the green horizontal arrows pointing toward the 3′ end. iii . Both filaments pair with the sequence matched regions in Copy 2 and form sequence matched heteroduplex products that extend to the 3′ ends of the initiating strands. DNA synthesis then completes two dsDNAs. The sequence region that includes the heteroduplex products and the newly synthesized DNA is shown in purple. The completion of the dsDNA is followed by Holliday junction resolution that is a crossover on the right side. As a result, the upper dsDNA lacks the sequence region between the two copies, and the lower dsDNA has two copies of the region between the two repeats. The duplicated regions are highlighted by the yellow rectangles. ( C ) Schematic of a DSB that occurs in Copy 1 of a repeated sequence. The appropriately oriented Chi sites are indicated by the bright green triangles, and the direction of RecBCD is shown by the black arrows. Copy 1 only includes appropriately oriented Chi sites in the dark red strand, so only the dark red strand can terminate in a region of Copy 1. ( D ) Same as C), but the DSB occurs to the left of Copy 1, which changes the number of correctly oriented Chi site in this region. The light pink filament cannot include a region of Copy 1 because RecBCD moves away from the DSB. ( E ) In a different sequence than C), Copy 1 includes appropriately oriented Chi sites on both strands, allowing the rearrangement shown in B); however, this never occurs in any of the 12 enteric bacteria we studied in this work.

    Techniques Used: Sequencing, DNA Synthesis, Synthesized

    DNA synthesis is required for a DNA polymerase to stabilize strand exchange products. ( A ) Schematic for experiments with M 3 ′ values of 0, 3, 5. ( B ) Graphic representation of the change in fluorescence (Δ F ) versus time curves from single trial experiments performed with dATP–ssDNA–RecA filaments and DNA Pol IV in which the blue, pink, and light-blue curves correspond to M 3′ values of 0, 3 and 5 base mismatches, respectively. Δ F is calculated as the difference between the measured fluorescence and the average initial fluorescence for heterologous dsDNA. ( C ) Analogous experiments as (B), but with LF-Bsu polymerase instead of DNA Pol IV.
    Figure Legend Snippet: DNA synthesis is required for a DNA polymerase to stabilize strand exchange products. ( A ) Schematic for experiments with M 3 ′ values of 0, 3, 5. ( B ) Graphic representation of the change in fluorescence (Δ F ) versus time curves from single trial experiments performed with dATP–ssDNA–RecA filaments and DNA Pol IV in which the blue, pink, and light-blue curves correspond to M 3′ values of 0, 3 and 5 base mismatches, respectively. Δ F is calculated as the difference between the measured fluorescence and the average initial fluorescence for heterologous dsDNA. ( C ) Analogous experiments as (B), but with LF-Bsu polymerase instead of DNA Pol IV.

    Techniques Used: DNA Synthesis, Fluorescence

    The presence of a second filament rescues instability caused by an ssDNA outgoing strand and a long homoduplex dsDNA (180 bp) that extends beyond the 3′ end of a filament. ( A ) Schematic of experiments with 66 bp of homoduplex dsDNA on the 3′ end of the filament and N values of 5, 20, 50 and 82 bp. ( B ) Graphic representation of the change in fluorescence (ΔΔ F ) versus time curves of the experiment represented in (A) with dATP–ssDNA–RecA filaments and DNA Pol IV. ΔΔ F is calculated as the difference between the measured fluorescence and the fluorescence for N = 5. The purple, red, and black curves correspond to N 2 = 82, 50 and 20 nt, respectively. ( C ) Schematic for experiments performed involving two filaments. In all of these experiments N 1 = 42. ( D ) Graphic representation of the change in fluorescence ΔΔ F versus time curves for single trials of the experiment represented in (C) with dATP–ssDNA–RecA filaments and DNA Pol IV. ΔΔ F was calculated as indicated above. Different N 2 values are represented with different curve colors, where purple, red and black correspond to N 2 = 82, 50 and 20 nt, respectively.
    Figure Legend Snippet: The presence of a second filament rescues instability caused by an ssDNA outgoing strand and a long homoduplex dsDNA (180 bp) that extends beyond the 3′ end of a filament. ( A ) Schematic of experiments with 66 bp of homoduplex dsDNA on the 3′ end of the filament and N values of 5, 20, 50 and 82 bp. ( B ) Graphic representation of the change in fluorescence (ΔΔ F ) versus time curves of the experiment represented in (A) with dATP–ssDNA–RecA filaments and DNA Pol IV. ΔΔ F is calculated as the difference between the measured fluorescence and the fluorescence for N = 5. The purple, red, and black curves correspond to N 2 = 82, 50 and 20 nt, respectively. ( C ) Schematic for experiments performed involving two filaments. In all of these experiments N 1 = 42. ( D ) Graphic representation of the change in fluorescence ΔΔ F versus time curves for single trials of the experiment represented in (C) with dATP–ssDNA–RecA filaments and DNA Pol IV. ΔΔ F was calculated as indicated above. Different N 2 values are represented with different curve colors, where purple, red and black correspond to N 2 = 82, 50 and 20 nt, respectively.

    Techniques Used: Fluorescence

    DNA synthesis stabilizes DSB repair occurring at a repeat. ( A ) Experimental schematic showing a typical ssDNA–RecA filament (orange line with blue ellipses) and 90 bp dsDNA. Δ L = D label – D init = 5 bp. The labeled dsDNA used in all of the experiments was the same, so each N value corresponds to a different filament sequence. For each N value, the green brackets highlight the green regions of the dsDNA that are homologous to the N bases at the 3′ end of the ssDNA. The other bases in the dsDNA are heterologous to the initiating ssDNA. The yellow region indicates D init = 15 bp. The remaining dsDNA is shown in magenta. The red circle and black star represent the rhodamine and fluorescein labels, respectively. They are positioned on the complementary (purple line) and outgoing (blue line) strands, respectively. ( B ) Graph representing the average over three trials of the change in fluorescence (Δ F ) versus time curves in experiments with dATP–ssDNA–RecA filaments and DNA Pol IV represented in A) for N = 75 (dark blue), 50 (red), 36 (gray), 20 (black), and heterologous filament (light blue). Δ F in counts per second (cps) is calculated as the difference between the measured fluorescence and the average initial fluorescence for heterologous dsDNA. The error bars show the standard deviation based on three trials. ( C ) Same as B) in the presence of ATP–ssDNA–RecA filaments and LF-Bsu polymerase.
    Figure Legend Snippet: DNA synthesis stabilizes DSB repair occurring at a repeat. ( A ) Experimental schematic showing a typical ssDNA–RecA filament (orange line with blue ellipses) and 90 bp dsDNA. Δ L = D label – D init = 5 bp. The labeled dsDNA used in all of the experiments was the same, so each N value corresponds to a different filament sequence. For each N value, the green brackets highlight the green regions of the dsDNA that are homologous to the N bases at the 3′ end of the ssDNA. The other bases in the dsDNA are heterologous to the initiating ssDNA. The yellow region indicates D init = 15 bp. The remaining dsDNA is shown in magenta. The red circle and black star represent the rhodamine and fluorescein labels, respectively. They are positioned on the complementary (purple line) and outgoing (blue line) strands, respectively. ( B ) Graph representing the average over three trials of the change in fluorescence (Δ F ) versus time curves in experiments with dATP–ssDNA–RecA filaments and DNA Pol IV represented in A) for N = 75 (dark blue), 50 (red), 36 (gray), 20 (black), and heterologous filament (light blue). Δ F in counts per second (cps) is calculated as the difference between the measured fluorescence and the average initial fluorescence for heterologous dsDNA. The error bars show the standard deviation based on three trials. ( C ) Same as B) in the presence of ATP–ssDNA–RecA filaments and LF-Bsu polymerase.

    Techniques Used: DNA Synthesis, Labeling, Sequencing, Fluorescence, Standard Deviation

    ΔΔ F versus time curves obtained with ATP–ssDNA–RecA filaments, LF-Bsu polymerase, and 180 bp labeled dsDNA construct. The purple, red and black curves correspond to N = 82, 50 and 20, respectively. ( A ) ΔΔ F versus time curves for one filament experiments with LF-Bsu, represented by the schematic shown in Figure 5A and after subtracting the heterologous DNA curve shown in Supplementary Figure S5A . ( B ) ΔΔ F versus time curves for two filament experiments in the presence of LF-Bsu, represented by the schematic shown in Figure 5C and after subtracting the heterologous DNA curve shown in Supplementary Figure S5B .
    Figure Legend Snippet: ΔΔ F versus time curves obtained with ATP–ssDNA–RecA filaments, LF-Bsu polymerase, and 180 bp labeled dsDNA construct. The purple, red and black curves correspond to N = 82, 50 and 20, respectively. ( A ) ΔΔ F versus time curves for one filament experiments with LF-Bsu, represented by the schematic shown in Figure 5A and after subtracting the heterologous DNA curve shown in Supplementary Figure S5A . ( B ) ΔΔ F versus time curves for two filament experiments in the presence of LF-Bsu, represented by the schematic shown in Figure 5C and after subtracting the heterologous DNA curve shown in Supplementary Figure S5B .

    Techniques Used: Labeling, Construct

    4) Product Images from "RecA homology search is promoted by mechanical stress along the scanned duplex DNA"

    Article Title: RecA homology search is promoted by mechanical stress along the scanned duplex DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr855

    Non-homologous binding is stabilized by external force. Preformed RecA–ssDNA filaments ( A–D ) or free RecA ( E–H ) added to λ dsDNAs. After 100 s at 57 pN, force was reduced (arrow) by 10 to 47 pN ( A, B, E, F ) or 40 to 17 pN ( C, D, G, H ). Non-homologous RecA–ssDNA binding is rapidly lost if force is reduced, irrespective of ATP hydrolysis ( A, D ). RecA binding (via site I) continues in ATPγS ( F, H ) and is lost in ATP if force is reduced
    Figure Legend Snippet: Non-homologous binding is stabilized by external force. Preformed RecA–ssDNA filaments ( A–D ) or free RecA ( E–H ) added to λ dsDNAs. After 100 s at 57 pN, force was reduced (arrow) by 10 to 47 pN ( A, B, E, F ) or 40 to 17 pN ( C, D, G, H ). Non-homologous RecA–ssDNA binding is rapidly lost if force is reduced, irrespective of ATP hydrolysis ( A, D ). RecA binding (via site I) continues in ATPγS ( F, H ) and is lost in ATP if force is reduced

    Techniques Used: Binding Assay

    Single molecule extension profiles for non-homologous RecA–ssDNA (1 kb) filaments binding when force is applied to the 3′5′-ends of one of the constituent strands in RecA buffer pH 7.6 containing ATPγS. ( A ) Force applied to the same constituent strand as in Figure 3 . Green curve: 55.7 pN, red: 59.1 pN, and purple: 58.4 pN. Control curves are black (56.6 pN), dark gray (59.8 pN) and light gray (57.5 pN). ( B ) Force was applied to the 3′5′-ends of the other constituent strand. Green: 56 pN, red: 59.6 pN, and purple: 58.4 pN. Control curves are light gray (59 pN) and dark gray (57.5 pN).
    Figure Legend Snippet: Single molecule extension profiles for non-homologous RecA–ssDNA (1 kb) filaments binding when force is applied to the 3′5′-ends of one of the constituent strands in RecA buffer pH 7.6 containing ATPγS. ( A ) Force applied to the same constituent strand as in Figure 3 . Green curve: 55.7 pN, red: 59.1 pN, and purple: 58.4 pN. Control curves are black (56.6 pN), dark gray (59.8 pN) and light gray (57.5 pN). ( B ) Force was applied to the 3′5′-ends of the other constituent strand. Green: 56 pN, red: 59.6 pN, and purple: 58.4 pN. Control curves are light gray (59 pN) and dark gray (57.5 pN).

    Techniques Used: Binding Assay

    Binding of RecA–ssDNA filaments to dsDNA pulled from different ends. ( A ) Δ L RecA versus force for 1000-nt filaments (magenta circles). λ dsDNAs are pulled on (i) 3′5′-ends of one strand, (ii) 3′3′-ends, (iii) 5′5′-ends, (iv) both ends of each of the two constituent strands of the dsDNA, respectively, and controls for each pulling technique in the absence of RecA (gray triangles). ( B ) Histograms of the fraction of the total number of beads that showed a particular Δ L RecA after a force has been applied for 60 s (magenta) for dsDNA pulled (from left to right) from 3′5′-ends of one strand, 3′3′ends, 5′5′-ends, and both ends of each of the two constituent strands of the dsDNA; force range: 52-55 pN and negative controls shown in gray. ( C ) Histograms of the fraction of the total number of beads that showed a particular Δ L RecA after a force has been applied for 60 s (magenta) for dsDNA pulled (from left to right) from 3′5′-ends of one strand, 3′3′-ends, 5′5′-ends and both ends of each of the two constituent strands of the dsDNA: force range: 52–57 pN; negative controls shown in gray. ( D ) Bar graphs for Δ L RecA values between 52 and 55 pN and 52 and 57 pN, for 1000-nt filaments. λ dsDNAs are pulled 3′5′-, 3′3′-, 5′5′- and both ends of both strands: positives (magenta) and controls (gray). In these experiments filaments were prepared in a buffer containing ATPγS and measured in a buffer containing ATPγS.
    Figure Legend Snippet: Binding of RecA–ssDNA filaments to dsDNA pulled from different ends. ( A ) Δ L RecA versus force for 1000-nt filaments (magenta circles). λ dsDNAs are pulled on (i) 3′5′-ends of one strand, (ii) 3′3′-ends, (iii) 5′5′-ends, (iv) both ends of each of the two constituent strands of the dsDNA, respectively, and controls for each pulling technique in the absence of RecA (gray triangles). ( B ) Histograms of the fraction of the total number of beads that showed a particular Δ L RecA after a force has been applied for 60 s (magenta) for dsDNA pulled (from left to right) from 3′5′-ends of one strand, 3′3′ends, 5′5′-ends, and both ends of each of the two constituent strands of the dsDNA; force range: 52-55 pN and negative controls shown in gray. ( C ) Histograms of the fraction of the total number of beads that showed a particular Δ L RecA after a force has been applied for 60 s (magenta) for dsDNA pulled (from left to right) from 3′5′-ends of one strand, 3′3′-ends, 5′5′-ends and both ends of each of the two constituent strands of the dsDNA: force range: 52–57 pN; negative controls shown in gray. ( D ) Bar graphs for Δ L RecA values between 52 and 55 pN and 52 and 57 pN, for 1000-nt filaments. λ dsDNAs are pulled 3′5′-, 3′3′-, 5′5′- and both ends of both strands: positives (magenta) and controls (gray). In these experiments filaments were prepared in a buffer containing ATPγS and measured in a buffer containing ATPγS.

    Techniques Used: Binding Assay

    RecA–ssDNA filaments extend non-homologous dsDNA pulled by 3′5′ ends. ( A ) Δ L RecA 's for λ dsDNAs controls with free ssDNA or λ dsDNA only (red, N = 87) and λ dsDNAs with RecA–ssDNA filaments (blue, N = 454). ( B ) Single molecule extension profiles for non-homologous binding. Different colors are used to represent the response for each different single molecule. The curves ending at positions corresponding to 1, 3 and 4 filaments are 57.0, 59.0 and 60.9 pN, respectively. The red and green curves ending at two-filament lengths correspond to 58.9 and 57.2 pN, respectively. The filaments were prepared in a buffer containing ATPγS, but measured in a buffer containing ATP Pauses at full filament lengths are indicated by horizontal bars the same color as the curve. ( C ) Δ L RecA probability distributions for periods of ≥2 s constant λ dsDNA length, for all filaments of 400–800 nt at 50–62 pN. ( D ) DNA extensions within homology search complexes on dsDNA (color) or λ ssDNA.
    Figure Legend Snippet: RecA–ssDNA filaments extend non-homologous dsDNA pulled by 3′5′ ends. ( A ) Δ L RecA 's for λ dsDNAs controls with free ssDNA or λ dsDNA only (red, N = 87) and λ dsDNAs with RecA–ssDNA filaments (blue, N = 454). ( B ) Single molecule extension profiles for non-homologous binding. Different colors are used to represent the response for each different single molecule. The curves ending at positions corresponding to 1, 3 and 4 filaments are 57.0, 59.0 and 60.9 pN, respectively. The red and green curves ending at two-filament lengths correspond to 58.9 and 57.2 pN, respectively. The filaments were prepared in a buffer containing ATPγS, but measured in a buffer containing ATP Pauses at full filament lengths are indicated by horizontal bars the same color as the curve. ( C ) Δ L RecA probability distributions for periods of ≥2 s constant λ dsDNA length, for all filaments of 400–800 nt at 50–62 pN. ( D ) DNA extensions within homology search complexes on dsDNA (color) or λ ssDNA.

    Techniques Used: Binding Assay

    5) Product Images from "ATP hydrolysis provides functions that promote rejection of pairings between different copies of long repeated sequences"

    Article Title: ATP hydrolysis provides functions that promote rejection of pairings between different copies of long repeated sequences

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx582

    Progression of strand exchange for different N = L dsDNAs. ( A ) Schematic representation of experiments that measure the fluorescence change due to the separation between the fluorescein (star) labeled complementary strand (purple line) and rhodamine (red circle) labeled outgoing strand (blue line). Base-pairing is indicated in yellow. The initiating strand and the RecA monomers are illustrated by the orange line and grey ovals, respectively. The big black and green stars represent quenched and unquenched fluorescence. ( B ) Δ F versus time curves for 98 nt filaments and dsDNAs of increasing length in ATPγS showing the change in cps from the initial value measured for a solution containing each dsDNA in the absence of ssDNA-RecA filaments; N = 15 (black), 20 (gray), 50 (red) and 75 (blue) bp. ( C ) Same as (B) but in the presence of ATP. The y-axis label for Figure 1B also applies to Figure 1C . ( D ) Each value of the change in fluorescence in ATPγS was subtracted to the final value averaged over the last 10 s; the inset is the same data on a logarithmic y-scale. ( E ) Same as (D) but in ATP. The y-axis label for Figure 1D also applies to Figure 1E .
    Figure Legend Snippet: Progression of strand exchange for different N = L dsDNAs. ( A ) Schematic representation of experiments that measure the fluorescence change due to the separation between the fluorescein (star) labeled complementary strand (purple line) and rhodamine (red circle) labeled outgoing strand (blue line). Base-pairing is indicated in yellow. The initiating strand and the RecA monomers are illustrated by the orange line and grey ovals, respectively. The big black and green stars represent quenched and unquenched fluorescence. ( B ) Δ F versus time curves for 98 nt filaments and dsDNAs of increasing length in ATPγS showing the change in cps from the initial value measured for a solution containing each dsDNA in the absence of ssDNA-RecA filaments; N = 15 (black), 20 (gray), 50 (red) and 75 (blue) bp. ( C ) Same as (B) but in the presence of ATP. The y-axis label for Figure 1B also applies to Figure 1C . ( D ) Each value of the change in fluorescence in ATPγS was subtracted to the final value averaged over the last 10 s; the inset is the same data on a logarithmic y-scale. ( E ) Same as (D) but in ATP. The y-axis label for Figure 1D also applies to Figure 1E .

    Techniques Used: Fluorescence, Labeling

    Results with experiments with fluorescent labels at various positions along the dsDNA. ( A ) Schematic representation of the 90 bp dsDNAs showing the position of the internal labels fluorescein (F) and rhodamine (Rho). ( B ) Normalized Δ F versus time curves for 98 nt ssDNA-RecA filament, 90 bp dsDNAs with internal labels at position d , and ATP; orange, green, blue, and purple lines correspond to labels d = 10, 20, 36 and 56, respectively. The inset shows the fit obtained with a simplified model (dashed lines); for details, see Supplementary Data . ( C ) Same as (B) for 75 nt ssDNA-RecA filament. ( D ) Total fluorescence for 98 nt ssDNA-RecA filament, 90 bp dsDNA containing internal labels at position d , and ATP; orange and blue lines correspond to labels d = 10 (average of five curves) and 36 (average of three curves). ( E ) Total fluorescence for 75 nt ssDNA-RecA filament, 90 bp dsDNA containing internal labels at position d , and ATP; orange and blue lines correspond to labels d = 10 (average of three curves) and 36 (average of three curves).
    Figure Legend Snippet: Results with experiments with fluorescent labels at various positions along the dsDNA. ( A ) Schematic representation of the 90 bp dsDNAs showing the position of the internal labels fluorescein (F) and rhodamine (Rho). ( B ) Normalized Δ F versus time curves for 98 nt ssDNA-RecA filament, 90 bp dsDNAs with internal labels at position d , and ATP; orange, green, blue, and purple lines correspond to labels d = 10, 20, 36 and 56, respectively. The inset shows the fit obtained with a simplified model (dashed lines); for details, see Supplementary Data . ( C ) Same as (B) for 75 nt ssDNA-RecA filament. ( D ) Total fluorescence for 98 nt ssDNA-RecA filament, 90 bp dsDNA containing internal labels at position d , and ATP; orange and blue lines correspond to labels d = 10 (average of five curves) and 36 (average of three curves). ( E ) Total fluorescence for 75 nt ssDNA-RecA filament, 90 bp dsDNA containing internal labels at position d , and ATP; orange and blue lines correspond to labels d = 10 (average of three curves) and 36 (average of three curves).

    Techniques Used: Fluorescence

    6) Product Images from "RADA is the main branch migration factor in plant mitochondrial recombination and its defect leads to mtDNA instability and cell cycle arrest"

    Article Title: RADA is the main branch migration factor in plant mitochondrial recombination and its defect leads to mtDNA instability and cell cycle arrest

    Journal: bioRxiv

    doi: 10.1101/856716

    DNA-binding and branch-migration activities of RADA. ( A ) EMSA experiments showing that RADA binds any ssDNA-containing DNA structure with higher affinity than dsDNA. Lower and higher molecular weigh complexes are indicated by white and black arrowheads respectively ( B ) Analysis on low concentration gel (4.5 % as compared to 8 % in A) of the formation of a high-molecular weight RADA filament on ssDNA, which is promoted by ATP or ADP (1 mM). The K201A mutant protein binds with equivalent affinity as the WT protein. Increasing concentrations of RADA used in A and B are indicated by the grey triangles. ( C ) In a in vitro strand-invasion reaction plant RADA accelerates branch-migration of DNA heteroduplexes initiated by RecA. An explanation of the different substrates and products is shown below the gel. ( D ) Ratio of final product as compared to the initial linear dsDNA substrate in 6 independent experiments, showing that in the presence of RADA there is faster resolution of branched intermediates. ( E ) RADA can alone finalize branch-migration initiated by RecA: a reaction at T=7 min was arrested by deproteination (left panel) and the DNA purified. Without addition of RecA or RADA there is no spontaneous progression of the reaction (middle panel), but RADA alone can resolve intermediates into the final product (right panel). ( F ) RADA alone cannot initiate strand invasion. ( G ) Mutation of the ATPase Walker domain of RADA (K201A) inhibits the reaction.
    Figure Legend Snippet: DNA-binding and branch-migration activities of RADA. ( A ) EMSA experiments showing that RADA binds any ssDNA-containing DNA structure with higher affinity than dsDNA. Lower and higher molecular weigh complexes are indicated by white and black arrowheads respectively ( B ) Analysis on low concentration gel (4.5 % as compared to 8 % in A) of the formation of a high-molecular weight RADA filament on ssDNA, which is promoted by ATP or ADP (1 mM). The K201A mutant protein binds with equivalent affinity as the WT protein. Increasing concentrations of RADA used in A and B are indicated by the grey triangles. ( C ) In a in vitro strand-invasion reaction plant RADA accelerates branch-migration of DNA heteroduplexes initiated by RecA. An explanation of the different substrates and products is shown below the gel. ( D ) Ratio of final product as compared to the initial linear dsDNA substrate in 6 independent experiments, showing that in the presence of RADA there is faster resolution of branched intermediates. ( E ) RADA can alone finalize branch-migration initiated by RecA: a reaction at T=7 min was arrested by deproteination (left panel) and the DNA purified. Without addition of RecA or RADA there is no spontaneous progression of the reaction (middle panel), but RADA alone can resolve intermediates into the final product (right panel). ( F ) RADA alone cannot initiate strand invasion. ( G ) Mutation of the ATPase Walker domain of RADA (K201A) inhibits the reaction.

    Techniques Used: Binding Assay, Migration, Concentration Assay, Molecular Weight, Mutagenesis, In Vitro, Purification

    7) Product Images from "Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics"

    Article Title: Evaluation and improvement of isothermal amplification methods for point-of-need plant disease diagnostics

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0235216

    Effect of betaine, recombinase and nucleotide addition on LAMP amplification. ( A ) Different combinations of RecA and betaine were used in LAMP reactions containing 1 ng target DNA and 1 mM ATP and the time needed for each sample to reach the fluorescence threshold of 0.5 (T threshold ) in real-time LAMP was determined (n = 10). ( B ) RecA and/or ATP were added into reactions containing 0.4 M betaine. Reactions without RecA and ATP were used as control (n = 3). “+” and “-” below each bar indicated the present and absent of the component respectively. ( C ) Additional 1 mM ATP, dATP, dTTP, dCTP or dGTP was added into reactions containing 0.4 M betaine. Reactions without additional ATP and dNTP were used as controls. ( D ) Different amounts of ATP were added into reactions containing 0.4 M betaine (n = 3). To investigate interplay between dNTPs and Mg 2+ , 4.8 mM (dark blue), 8.8 mM (red) or 12.8 mM (green) total dNTPs was added into reactions containing 0.4 M betaine and either ( E ) 8 mM Mg 2+ or ( F ) 10 mM Mg 2+ respectively. ( G ) Two sets of reactions containing 0.4 M betaine and either 0 mM (black) or 2 mM (grey) ATP were prepared. Additional swarm and/or loop primers along with standard LAMP primers were added into each set of reactions. Standard LAMP primers alone were used as controls (n = 4). All T threshold data was analysed using one-way ANOVA with a post-hoc Tukey’s multiple comparison of means test (p value
    Figure Legend Snippet: Effect of betaine, recombinase and nucleotide addition on LAMP amplification. ( A ) Different combinations of RecA and betaine were used in LAMP reactions containing 1 ng target DNA and 1 mM ATP and the time needed for each sample to reach the fluorescence threshold of 0.5 (T threshold ) in real-time LAMP was determined (n = 10). ( B ) RecA and/or ATP were added into reactions containing 0.4 M betaine. Reactions without RecA and ATP were used as control (n = 3). “+” and “-” below each bar indicated the present and absent of the component respectively. ( C ) Additional 1 mM ATP, dATP, dTTP, dCTP or dGTP was added into reactions containing 0.4 M betaine. Reactions without additional ATP and dNTP were used as controls. ( D ) Different amounts of ATP were added into reactions containing 0.4 M betaine (n = 3). To investigate interplay between dNTPs and Mg 2+ , 4.8 mM (dark blue), 8.8 mM (red) or 12.8 mM (green) total dNTPs was added into reactions containing 0.4 M betaine and either ( E ) 8 mM Mg 2+ or ( F ) 10 mM Mg 2+ respectively. ( G ) Two sets of reactions containing 0.4 M betaine and either 0 mM (black) or 2 mM (grey) ATP were prepared. Additional swarm and/or loop primers along with standard LAMP primers were added into each set of reactions. Standard LAMP primers alone were used as controls (n = 4). All T threshold data was analysed using one-way ANOVA with a post-hoc Tukey’s multiple comparison of means test (p value

    Techniques Used: Amplification, Fluorescence, Significance Assay

    8) Product Images from "Simple and efficient purification of Escherichia coli DNA polymerase V: Cofactor requirements for optimal activity and processivity in vitro"

    Article Title: Simple and efficient purification of Escherichia coli DNA polymerase V: Cofactor requirements for optimal activity and processivity in vitro

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2012.01.012

    Processivity of pol V Mut in the presence or absence of β-clamp and SSB. (A) RecA was incubated with the biotinylated oligomers linked to streptavidin-coated agarose resin. Resulting RecA* was mixed with pol V to produce pol VMut which was recovered
    Figure Legend Snippet: Processivity of pol V Mut in the presence or absence of β-clamp and SSB. (A) RecA was incubated with the biotinylated oligomers linked to streptavidin-coated agarose resin. Resulting RecA* was mixed with pol V to produce pol VMut which was recovered

    Techniques Used: Incubation

    9) Product Images from "The poor homology stringency in the heteroduplex allows strand exchange to incorporate desirable mismatches without sacrificing recognition in vivo"

    Article Title: The poor homology stringency in the heteroduplex allows strand exchange to incorporate desirable mismatches without sacrificing recognition in vivo

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv610

    Molecular Dynamics simulation for poly dA-dT and poly dG-dT dsDNA in site I. ( A ) Schematic of the molecular dynamics simulation system. The solvation water is shown as the transparent box and the sodium and chloride ions are shown as pink and purple sphere, respectively. The molecule in surface representation is the RecA-DNA complex. ( B ) Structure of the matched and mismatched DNA in site I at the end of the 15 ns conventional MD simulation. The initiating, and matched and mismatched complementary strands are shown in yellow, cyan and green, respectively. The overlapping structures are obtained from the last 10 ns of the simulation with 1 ns gap. ( C and D ). Detail illustrations of the stable nucleotide triplet at the end of the simulation for matched and mismatched strands.
    Figure Legend Snippet: Molecular Dynamics simulation for poly dA-dT and poly dG-dT dsDNA in site I. ( A ) Schematic of the molecular dynamics simulation system. The solvation water is shown as the transparent box and the sodium and chloride ions are shown as pink and purple sphere, respectively. The molecule in surface representation is the RecA-DNA complex. ( B ) Structure of the matched and mismatched DNA in site I at the end of the 15 ns conventional MD simulation. The initiating, and matched and mismatched complementary strands are shown in yellow, cyan and green, respectively. The overlapping structures are obtained from the last 10 ns of the simulation with 1 ns gap. ( C and D ). Detail illustrations of the stable nucleotide triplet at the end of the simulation for matched and mismatched strands.

    Techniques Used:

    10) Product Images from "Complementary strand relocation may play vital roles in RecA-based homology recognition"

    Article Title: Complementary strand relocation may play vital roles in RecA-based homology recognition

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks769

    Proposed schematics of the strand exchange process. ( A ) Representation of the side view of the X-ray structure of the dsDNA heteroduplex in the final post-strand exchange state with incoming and complementary strands indicated by the cyan and red stick renderings. Orange, cyan and red arrows indicate the positions of the corresponding phosphates on the outgoing, incoming, and complementary strands in the final post-strand exchange structure. The VMD ( 9 ) renderings of RecA crystal structure 3CMX ( 8 ) show site II residues Arg226 (pink), Arg243 (yellow) and Lys245 (magenta) with charged nitrogen atoms (blue). The outgoing strand position was calculated by minimizing the energy of the interaction with those residues. The grey arrow points at the red plus sign indicating the proposed position of the corresponding complementary strand phosphate in the proposed intermediate structure. ( B ) Bottom view of the same structure. Circles correspond to the radii occupied by the phosphates. ( C ) Information as in Figure 1 B, but only one set of the corresponding phosphates is shown. Final state phosphate positions for the incoming, complementary, and outgoing strands are shown with filled colored circles. Paired bases shown as lines. The complementary strand position in the proposed intermediate structure is shown by the grey circle. ( D ) Same as Figure 1 B with circles and base pairs added to show radial positions of the corresponding phosphate groups and repositioning of the complementary strand.
    Figure Legend Snippet: Proposed schematics of the strand exchange process. ( A ) Representation of the side view of the X-ray structure of the dsDNA heteroduplex in the final post-strand exchange state with incoming and complementary strands indicated by the cyan and red stick renderings. Orange, cyan and red arrows indicate the positions of the corresponding phosphates on the outgoing, incoming, and complementary strands in the final post-strand exchange structure. The VMD ( 9 ) renderings of RecA crystal structure 3CMX ( 8 ) show site II residues Arg226 (pink), Arg243 (yellow) and Lys245 (magenta) with charged nitrogen atoms (blue). The outgoing strand position was calculated by minimizing the energy of the interaction with those residues. The grey arrow points at the red plus sign indicating the proposed position of the corresponding complementary strand phosphate in the proposed intermediate structure. ( B ) Bottom view of the same structure. Circles correspond to the radii occupied by the phosphates. ( C ) Information as in Figure 1 B, but only one set of the corresponding phosphates is shown. Final state phosphate positions for the incoming, complementary, and outgoing strands are shown with filled colored circles. Paired bases shown as lines. The complementary strand position in the proposed intermediate structure is shown by the grey circle. ( D ) Same as Figure 1 B with circles and base pairs added to show radial positions of the corresponding phosphate groups and repositioning of the complementary strand.

    Techniques Used:

    Proposed steps in strand exchange and free RecA binding. ( A ) Schematic of the steps involved in the proposed strand exchange process. Grey areas show regions occupied by the protein, excluding the L2 loop: ( i ) incoming strand (cyan), in the post-strand exchange state. ( ii ) dsDNA outgoing strand (orange circle) bound into Site II; complementary strand (red filled circle) in proposed intermediate position. ( iii ) Complementary strand bases and incoming strand bases rotate in search of homology, where an L2 loop may rotate with the incoming strand. ( iv ) Strand exchanged state that may benefit from an interaction with the L2 loop if the bases are homologous. ( v ) The heteroduplex dsDNA rotates to the final X-ray structure position, possibly accompanied by the L2 loop. ( B ) Schematic of possible steps in the binding of free RecA to dsDNA: ( i ) an additional free RecA binds to the dsDNA in the intermediate state, possibly accompanied by an interaction with the L2 loop. ( ii ) dsDNA (red circle) rotates to the final state shown by the x-ray structure, possibly accompanied by the L2 loop.
    Figure Legend Snippet: Proposed steps in strand exchange and free RecA binding. ( A ) Schematic of the steps involved in the proposed strand exchange process. Grey areas show regions occupied by the protein, excluding the L2 loop: ( i ) incoming strand (cyan), in the post-strand exchange state. ( ii ) dsDNA outgoing strand (orange circle) bound into Site II; complementary strand (red filled circle) in proposed intermediate position. ( iii ) Complementary strand bases and incoming strand bases rotate in search of homology, where an L2 loop may rotate with the incoming strand. ( iv ) Strand exchanged state that may benefit from an interaction with the L2 loop if the bases are homologous. ( v ) The heteroduplex dsDNA rotates to the final X-ray structure position, possibly accompanied by the L2 loop. ( B ) Schematic of possible steps in the binding of free RecA to dsDNA: ( i ) an additional free RecA binds to the dsDNA in the intermediate state, possibly accompanied by an interaction with the L2 loop. ( ii ) dsDNA (red circle) rotates to the final state shown by the x-ray structure, possibly accompanied by the L2 loop.

    Techniques Used: Binding Assay

    Comparison of the measured dsDNA elongation rates due to strand exchange and the binding of free RecA to dsDNA. ( A ) Arrhenius plot: strand exchange rates in bulk experiments and no external force ( 21 ) (black line); single-molecule strand exchange rates in ATP or ATPγS: grey diamonds ( 23 ) and grey squares ( 24 ); dsDNA release rate at the back of the strand exchange window in ATP: grey triangle ( 22 ); new results for strand exchange in ATPγS (red triangles), ATPγS–ADP (outlined-red square), and free RecA binding (red circles). Error bars: confidence intervals. ( B ) Extension rates as a function of MgCl 2 concentration. Log of single-molecule dsDNA extension rates (nm/s) as a function of MgCl 2 concentration (mM) for both free RecA binding (navy diamonds) and strand exchange (green triangles) in ATPγS at 22°C. MgCl 2 concentrations varied from 0.1 to 10 mM. The dependence of the rates of free RecA binding on MgCl 2 concentration is fit by a logarithmic trend-line (black line).
    Figure Legend Snippet: Comparison of the measured dsDNA elongation rates due to strand exchange and the binding of free RecA to dsDNA. ( A ) Arrhenius plot: strand exchange rates in bulk experiments and no external force ( 21 ) (black line); single-molecule strand exchange rates in ATP or ATPγS: grey diamonds ( 23 ) and grey squares ( 24 ); dsDNA release rate at the back of the strand exchange window in ATP: grey triangle ( 22 ); new results for strand exchange in ATPγS (red triangles), ATPγS–ADP (outlined-red square), and free RecA binding (red circles). Error bars: confidence intervals. ( B ) Extension rates as a function of MgCl 2 concentration. Log of single-molecule dsDNA extension rates (nm/s) as a function of MgCl 2 concentration (mM) for both free RecA binding (navy diamonds) and strand exchange (green triangles) in ATPγS at 22°C. MgCl 2 concentrations varied from 0.1 to 10 mM. The dependence of the rates of free RecA binding on MgCl 2 concentration is fit by a logarithmic trend-line (black line).

    Techniques Used: Binding Assay, Concentration Assay

    Effect of force applied to different ends of dsDNA λ-phage during strand exchange and free RecA binding. ( A ) Elongation rate histogram for strand exchange in ATPγS at 30°C; peak 1 shows the characteristic rate of 211.8 bp/min (0.60 nm/s) whereas peak 0 corresponds to molecules that were followed but showed no change in extension. ( B ) Extension rates as a function of temperature for different 3′5′- and 3′3′-pulling techniques. Arrhenius plot of single-molecule extension rates as a function of temperature for free RecA binding and strand exchange in ATPγS and 10 mM MgCl 2 with 3′5′ and 3′3′ pulling techniques. Strand exchange rates in bulk experiments and no external force ( 21 ) (grey line); free RecA binding while pulling 3′5′ from the complementary strand (red plus signs); free RecA binding while pulling 3′5′ from the outgoing strand (blue × symbols). Strand exchange rate while pulling 3′5′ from the complementary strand (red squares) and while pulling the other 3′5′ strand with the alternative filament complementary to the pulled strand (red diamond). Strand exchange rate while pulling 3′5′ from the outgoing strand (blue circles); strand exchange rate while pulling 3′3′ from the outgoing strand nearest the filament (grey triangle) and strand exchange rate while pulling 3′3′ from the complementary strand nearest the filament (grey upside-down triangle). Error bars: confidence intervals. ( C ) Schematic representation of the effect of force applied to different ends of the dsDNA constructs during strand exchange experiments. ( i , ii and iii ) dsDNA pulled from 3′5′-ends with stressed and unstressed base pairs shown in magenta and blue, respectively. ( iv and v ) dsDNA pulled from the 3′3′-ends with stressed and unstressed base pairs shown in magenta and blue, respectively. The grey and black ssDNA correspond to filaments complementary to opposite strands of the dsDNA. In the representation of strand exchange in the first row, the RecA molecules were omitted for better clarity. The lavender band indicates the GC-rich end in λ-phage dsDNA. The ellipses in the second row indicate RecA monomers with Site I and Site II shown in grey and purple, respectively. The outgoing, complementary, and incoming strands are shown in orange, red and cyan, respectively. The effect of pulling the 3′5′-ends of the complementary strand, 3′5′-ends of the outgoing strand, and 3′3′-ends of the dsDNA is represented. The symbols under each figure correspond to the symbols in Figure 3 B.
    Figure Legend Snippet: Effect of force applied to different ends of dsDNA λ-phage during strand exchange and free RecA binding. ( A ) Elongation rate histogram for strand exchange in ATPγS at 30°C; peak 1 shows the characteristic rate of 211.8 bp/min (0.60 nm/s) whereas peak 0 corresponds to molecules that were followed but showed no change in extension. ( B ) Extension rates as a function of temperature for different 3′5′- and 3′3′-pulling techniques. Arrhenius plot of single-molecule extension rates as a function of temperature for free RecA binding and strand exchange in ATPγS and 10 mM MgCl 2 with 3′5′ and 3′3′ pulling techniques. Strand exchange rates in bulk experiments and no external force ( 21 ) (grey line); free RecA binding while pulling 3′5′ from the complementary strand (red plus signs); free RecA binding while pulling 3′5′ from the outgoing strand (blue × symbols). Strand exchange rate while pulling 3′5′ from the complementary strand (red squares) and while pulling the other 3′5′ strand with the alternative filament complementary to the pulled strand (red diamond). Strand exchange rate while pulling 3′5′ from the outgoing strand (blue circles); strand exchange rate while pulling 3′3′ from the outgoing strand nearest the filament (grey triangle) and strand exchange rate while pulling 3′3′ from the complementary strand nearest the filament (grey upside-down triangle). Error bars: confidence intervals. ( C ) Schematic representation of the effect of force applied to different ends of the dsDNA constructs during strand exchange experiments. ( i , ii and iii ) dsDNA pulled from 3′5′-ends with stressed and unstressed base pairs shown in magenta and blue, respectively. ( iv and v ) dsDNA pulled from the 3′3′-ends with stressed and unstressed base pairs shown in magenta and blue, respectively. The grey and black ssDNA correspond to filaments complementary to opposite strands of the dsDNA. In the representation of strand exchange in the first row, the RecA molecules were omitted for better clarity. The lavender band indicates the GC-rich end in λ-phage dsDNA. The ellipses in the second row indicate RecA monomers with Site I and Site II shown in grey and purple, respectively. The outgoing, complementary, and incoming strands are shown in orange, red and cyan, respectively. The effect of pulling the 3′5′-ends of the complementary strand, 3′5′-ends of the outgoing strand, and 3′3′-ends of the dsDNA is represented. The symbols under each figure correspond to the symbols in Figure 3 B.

    Techniques Used: Binding Assay, Construct

    11) Product Images from "Single-molecule studies of the stringency factors and rates governing the polymerization of RecA on double-stranded DNA"

    Article Title: Single-molecule studies of the stringency factors and rates governing the polymerization of RecA on double-stranded DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr013

    Minimum force required to observe a decrease in length versus time. ( A ) 1 mM ATP at pH 7.6 and several RecA concentrations; the inset shows the change in length after changing force to 5 pN in 4 µM RecA, 1 mM ATP and pH 7.6. ( B ) 1 µM RecA at pH 7.6, and several ATP concentrations.
    Figure Legend Snippet: Minimum force required to observe a decrease in length versus time. ( A ) 1 mM ATP at pH 7.6 and several RecA concentrations; the inset shows the change in length after changing force to 5 pN in 4 µM RecA, 1 mM ATP and pH 7.6. ( B ) 1 µM RecA at pH 7.6, and several ATP concentrations.

    Techniques Used:

    Fluorescent images of samples incubated in the presence of 1 µM-labeled RecA for 4 h at 37°C and 0.1 µM YOYO for 2 min. ( A ) 1 mM ATP; ( B ) 1 mM ATPγS.
    Figure Legend Snippet: Fluorescent images of samples incubated in the presence of 1 µM-labeled RecA for 4 h at 37°C and 0.1 µM YOYO for 2 min. ( A ) 1 mM ATP; ( B ) 1 mM ATPγS.

    Techniques Used: Incubation, Labeling

    Extension versus time for RecA growth on dsDNA at pH 7.6, 10 mM MgCl 2 , and 1 mM ATP at 10 pN, 30 pN, 40 pN and 50 pN (bottom curve to top curve, respectively). ( A ) Full-time range. ( B ) Replotting the first 100 s where a few growth rates that are several times higher than the minimum rate are indicated with the black lines next to each curve. A few lines labeled as 0× show absence of growth at the lowest forces or pauses at higher forces.
    Figure Legend Snippet: Extension versus time for RecA growth on dsDNA at pH 7.6, 10 mM MgCl 2 , and 1 mM ATP at 10 pN, 30 pN, 40 pN and 50 pN (bottom curve to top curve, respectively). ( A ) Full-time range. ( B ) Replotting the first 100 s where a few growth rates that are several times higher than the minimum rate are indicated with the black lines next to each curve. A few lines labeled as 0× show absence of growth at the lowest forces or pauses at higher forces.

    Techniques Used: Labeling

    RecA polymerization in the absence of hydrolysis or negligible hydrolysis: elongation of one single dsDNA molecule in 1 mM ATP and 10 mM CaCl 2 , pH 7.6 at 21 pN, 32 pN, 44 pN and 54 pN (bottom curve to top curve, respectively).
    Figure Legend Snippet: RecA polymerization in the absence of hydrolysis or negligible hydrolysis: elongation of one single dsDNA molecule in 1 mM ATP and 10 mM CaCl 2 , pH 7.6 at 21 pN, 32 pN, 44 pN and 54 pN (bottom curve to top curve, respectively).

    Techniques Used:

    Nucleation through overstretching. ( A ) Overstretching curve for lambda-phage dsDNA. ( B ) Absence of growth without overstretching and constant force of 50 pN (black curve); however, a fast increase in force through the overstretching transition (sharp peak) can start RecA polymerization showing linear growth at 50 pN on dsDNA at pH 7.6, 10 mM MgCl 2 , 1 mM ATP and 1 µM RecA (gray and black curve). A rapid change in force between 47 and 55 pN is indicated by the arrow.
    Figure Legend Snippet: Nucleation through overstretching. ( A ) Overstretching curve for lambda-phage dsDNA. ( B ) Absence of growth without overstretching and constant force of 50 pN (black curve); however, a fast increase in force through the overstretching transition (sharp peak) can start RecA polymerization showing linear growth at 50 pN on dsDNA at pH 7.6, 10 mM MgCl 2 , 1 mM ATP and 1 µM RecA (gray and black curve). A rapid change in force between 47 and 55 pN is indicated by the arrow.

    Techniques Used:

    Histogrammed slopes of extension versus time obtained for several experiments where the elongation at constant forces between 20 and 40 pN was measured in 1 µM RecA, 1 mM ATPγS and 10 mM MgCl 2 at pH 7.6. ( A ) Slopes at times > 1000 s at constant force, where the dominant peak at 0.48 nm/s (1×) differs from the average by 2 SDs. ( B ) Slopes at times 10–400 s at constant force, where arrows mark slopes at 1×, 2×, 3×, 4×, 6× and 10× integer multiples of the dominant rate. ( C ) Slopes of extension versus time measured in 1 mM ATP and 10 mM CaCl 2 , showing the 1×, 2× and 3× multiples of the dominant rate of 0.33 nm/s, 0.010 nm/s bin size; ( D ) 0.050 nm/s bin size; ( E ) 0.165 nm/s bin size, where the arrows show the integer multiples of the 1× rate.
    Figure Legend Snippet: Histogrammed slopes of extension versus time obtained for several experiments where the elongation at constant forces between 20 and 40 pN was measured in 1 µM RecA, 1 mM ATPγS and 10 mM MgCl 2 at pH 7.6. ( A ) Slopes at times > 1000 s at constant force, where the dominant peak at 0.48 nm/s (1×) differs from the average by 2 SDs. ( B ) Slopes at times 10–400 s at constant force, where arrows mark slopes at 1×, 2×, 3×, 4×, 6× and 10× integer multiples of the dominant rate. ( C ) Slopes of extension versus time measured in 1 mM ATP and 10 mM CaCl 2 , showing the 1×, 2× and 3× multiples of the dominant rate of 0.33 nm/s, 0.010 nm/s bin size; ( D ) 0.050 nm/s bin size; ( E ) 0.165 nm/s bin size, where the arrows show the integer multiples of the 1× rate.

    Techniques Used:

    12) Product Images from "Zinc blocks SOS-induced antibiotic resistance via inhibition of RecA in Escherichia coli"

    Article Title: Zinc blocks SOS-induced antibiotic resistance via inhibition of RecA in Escherichia coli

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0178303

    Regulation LexA by SOS activators and by zinc in E . coli . Panel A, immunoblot for LexA in whole-cell extracts of cultures of E . coli CP9 after a 3 h exposure to ciprofloxacin with and without zinc. Uncleaved LexA appeared to migrate in the form of a LexA dimer in these blots; while the cleaved LexA product ran at ~ 15 kDa. Panel B, densitometry scan of blot in Panel A, raw (left) and corrected for the effects of treatment on growth (right panel). Panel C, densitometry scan of a LexA blot (not shown) after a 1 h exposure to ciprofloxacin in Popeye-1. Panels D- H, RecA-mediated LexA cleavage assays in vitro, showing immunoblots against LexA. Purified LexA and RecA were incubated in vitro in the presence of absence of necessary cofactors, such as ssDNA and ATP or ATP- γ -S as described in the Methods section Panel D, RecA-mediated cleavage of LexA. An unlabeled lane to the left of lane 1 contained RecA alone, showing that the antibody does not cross-react between the two proteins. All the labeled lanes in Panel D received RecA, LexA, and a 38-mer oligonucleotide. Lane 1, no ATP; Lanes 2 and 3 also received 0.3 mM ATP; Lanes 4 and 5 also received 0.3 mM ATP- γ- S. Faint LexA cleavage products were visible in lanes 2–5 in the original blots, arrows ; Lanes 6 and 7, plus ATP- γ- S and 1 μM zinc acetate; Lanes 8 and 9, plus ATP- γ-S and 1 μM MnCl 2 ; Lane 10 received 0.3 mM GTP, which does not support RecA activation, as an additional control. Panel E, densitometry scan of the chemiluminescence signal from the blot shown in Panel D. Panel F, dose-response relationship of ATP- γ- S concentration vs. LexA cleavage in the absence and presence of 1 μM zinc acetate, showing protection by zinc against LexA cleavage at 0.1 to 0.3 mM ATP-γ - S. Panel G, combined results of 4 separate experiments testing for the effect of zinc acetate, and four experiments with MnCl 2 on LexA cleavage, with results normalized to the no- ATP- γ- S control so that separate experiments could be compared. Panel H, lack of protection by zinc on LexA auto-cleavage induced by incubation at pH 9. Control lanes 1 and 2 show LexA kept at pH 7.8; Lanes 3–6 show LexA protein incubated for 15 min at pH 9, 37°. Lanes 7–10 show samples incubated at pH 9 for 30 min, 37°. Lanes 5–6 and 9–10 also received 1 μM zinc acetate.
    Figure Legend Snippet: Regulation LexA by SOS activators and by zinc in E . coli . Panel A, immunoblot for LexA in whole-cell extracts of cultures of E . coli CP9 after a 3 h exposure to ciprofloxacin with and without zinc. Uncleaved LexA appeared to migrate in the form of a LexA dimer in these blots; while the cleaved LexA product ran at ~ 15 kDa. Panel B, densitometry scan of blot in Panel A, raw (left) and corrected for the effects of treatment on growth (right panel). Panel C, densitometry scan of a LexA blot (not shown) after a 1 h exposure to ciprofloxacin in Popeye-1. Panels D- H, RecA-mediated LexA cleavage assays in vitro, showing immunoblots against LexA. Purified LexA and RecA were incubated in vitro in the presence of absence of necessary cofactors, such as ssDNA and ATP or ATP- γ -S as described in the Methods section Panel D, RecA-mediated cleavage of LexA. An unlabeled lane to the left of lane 1 contained RecA alone, showing that the antibody does not cross-react between the two proteins. All the labeled lanes in Panel D received RecA, LexA, and a 38-mer oligonucleotide. Lane 1, no ATP; Lanes 2 and 3 also received 0.3 mM ATP; Lanes 4 and 5 also received 0.3 mM ATP- γ- S. Faint LexA cleavage products were visible in lanes 2–5 in the original blots, arrows ; Lanes 6 and 7, plus ATP- γ- S and 1 μM zinc acetate; Lanes 8 and 9, plus ATP- γ-S and 1 μM MnCl 2 ; Lane 10 received 0.3 mM GTP, which does not support RecA activation, as an additional control. Panel E, densitometry scan of the chemiluminescence signal from the blot shown in Panel D. Panel F, dose-response relationship of ATP- γ- S concentration vs. LexA cleavage in the absence and presence of 1 μM zinc acetate, showing protection by zinc against LexA cleavage at 0.1 to 0.3 mM ATP-γ - S. Panel G, combined results of 4 separate experiments testing for the effect of zinc acetate, and four experiments with MnCl 2 on LexA cleavage, with results normalized to the no- ATP- γ- S control so that separate experiments could be compared. Panel H, lack of protection by zinc on LexA auto-cleavage induced by incubation at pH 9. Control lanes 1 and 2 show LexA kept at pH 7.8; Lanes 3–6 show LexA protein incubated for 15 min at pH 9, 37°. Lanes 7–10 show samples incubated at pH 9 for 30 min, 37°. Lanes 5–6 and 9–10 also received 1 μM zinc acetate.

    Techniques Used: In Vitro, Western Blot, Purification, Incubation, Labeling, Activation Assay, Concentration Assay

    13) Product Images from "RADA is the main branch migration factor in plant mitochondrial recombination and its defect leads to mtDNA instability and cell cycle arrest"

    Article Title: RADA is the main branch migration factor in plant mitochondrial recombination and its defect leads to mtDNA instability and cell cycle arrest

    Journal: bioRxiv

    doi: 10.1101/856716

    DNA-binding and branch-migration activities of RADA. ( A ) EMSA experiments showing that RADA binds any ssDNA-containing DNA structure with higher affinity than dsDNA. Lower and higher molecular weigh complexes are indicated by white and black arrowheads respectively ( B ) Analysis on low concentration gel (4.5 % as compared to 8 % in A) of the formation of a high-molecular weight RADA filament on ssDNA, which is promoted by ATP or ADP (1 mM). The K201A mutant protein binds with equivalent affinity as the WT protein. Increasing concentrations of RADA used in A and B are indicated by the grey triangles. ( C ) In a in vitro strand-invasion reaction plant RADA accelerates branch-migration of DNA heteroduplexes initiated by RecA. An explanation of the different substrates and products is shown below the gel. ( D ) Ratio of final product as compared to the initial linear dsDNA substrate in 6 independent experiments, showing that in the presence of RADA there is faster resolution of branched intermediates. ( E ) RADA can alone finalize branch-migration initiated by RecA: a reaction at T=7 min was arrested by deproteination (left panel) and the DNA purified. Without addition of RecA or RADA there is no spontaneous progression of the reaction (middle panel), but RADA alone can resolve intermediates into the final product (right panel). ( F ) RADA alone cannot initiate strand invasion. ( G ) Mutation of the ATPase Walker domain of RADA (K201A) inhibits the reaction.
    Figure Legend Snippet: DNA-binding and branch-migration activities of RADA. ( A ) EMSA experiments showing that RADA binds any ssDNA-containing DNA structure with higher affinity than dsDNA. Lower and higher molecular weigh complexes are indicated by white and black arrowheads respectively ( B ) Analysis on low concentration gel (4.5 % as compared to 8 % in A) of the formation of a high-molecular weight RADA filament on ssDNA, which is promoted by ATP or ADP (1 mM). The K201A mutant protein binds with equivalent affinity as the WT protein. Increasing concentrations of RADA used in A and B are indicated by the grey triangles. ( C ) In a in vitro strand-invasion reaction plant RADA accelerates branch-migration of DNA heteroduplexes initiated by RecA. An explanation of the different substrates and products is shown below the gel. ( D ) Ratio of final product as compared to the initial linear dsDNA substrate in 6 independent experiments, showing that in the presence of RADA there is faster resolution of branched intermediates. ( E ) RADA can alone finalize branch-migration initiated by RecA: a reaction at T=7 min was arrested by deproteination (left panel) and the DNA purified. Without addition of RecA or RADA there is no spontaneous progression of the reaction (middle panel), but RADA alone can resolve intermediates into the final product (right panel). ( F ) RADA alone cannot initiate strand invasion. ( G ) Mutation of the ATPase Walker domain of RADA (K201A) inhibits the reaction.

    Techniques Used: Binding Assay, Migration, Concentration Assay, Molecular Weight, Mutagenesis, In Vitro, Purification

    Related Articles

    Transduction:

    Article Title: Mechanism of RecA-mediated homologous recombination revisited by single molecule nanomanipulation
    Article Snippet: .. Annu Rev Biochem 63 : 991–1043 [ ] Kowalczykowski SC, Krupp R (1995) DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions . ..

    Binding Assay:

    Article Title: Residues in the fingers domain of the translesion DNA polymerase DinB enable its unique participation in error-prone double-strand break repair
    Article Snippet: DinB(C66A) was purified from the TMCΔD strain (BL21-AI Δ dinB , Δ umuDC ( )) using an identical methodology. .. RecA nucleoprotein filament was formed by adding ssDNA (0.06 μ m ; and ), RecA (2 μ m ; New England Biolabs, Ipswich, MA), dATP (1 m m ), pyruvate kinase (10 units/ml), phosphoenolpyruvate (3 m m ), and single-stranded binding protein (0.2 μ m , Epicenter, Madison, WI) in RecA buffer (70 m m Tris-HCl, 10 m m MgCl2 , and 5 m m DTT, pH 7.6) at 37 °C for 10 min. ..

    Article Title: Effects of DNA sequence and structure on binding of RecA to single-stranded DNA
    Article Snippet: When our exponential distribution Dl is used, these two relations yield an expression for the beacon folding free energy Δ G f : With ɛ = 15.5 k B T measured for poly(T) (unpublished data) we obtain Δ G f = 4.3 k B T , which is smaller by a factor 2 than the folding computation. .. This paper addresses the issue of collective binding of RecA protein to DNA and the physical mechanisms that modulate the specificity of this interaction. .. We presented experiments of RecA polymerization on ssDNA oligomers using fluorescence anisotropy with improved resolution due to a microdispensing system.

    Article Title: The positioning of Chi sites allows the RecBCD pathway to suppress some genomic rearrangements
    Article Snippet: .. The filaments were initially prepared by incubating 0.06 μM ssDNA (final concentration ∼6 μM in bases) with 2 μM RecA (NEB) in the presence of 1 mM cofactor (ATP or dATP), 10 U/ml of pyruvate kinase, 3 mM phosphoenolpyruvate, and 0.2 μM single-stranded binding protein (SSB) (Epicentre) in RecA buffer (70 mM Tris–HCl, 10 mM MgCl2 and 5 mM dithiothreitol, pH 7.6) at 37°C for 10 min. FRET experiments followed the emission of the fluorescein label by using 493-nm excitation during 30 min; the emission was read as counts per second (cps) at 518 nm every one second. ..

    other:

    Article Title: Mechanism of RecA-mediated homologous recombination revisited by single molecule nanomanipulation
    Article Snippet: Biopolymers 42 : 383–385 Cox MM (2000) Recombinational DNA repair in bacteria and the RecA protein .

    Functional Assay:

    Article Title: Opposing Roles for Two Molecular Forms of Replication Protein A in Rad51-Rad54-Mediated DNA Recombination in Plasmodium falciparum
    Article Snippet: .. The assay mixture contained reaction buffer (25 mM Tris-HCl, pH 7.5, and 5% glycerol), 10 mM MgCl2 , 5 µM circular ΦX 174 RF I dsDNA (New England Biolabs [NEB]), 15 µM ΦX virion dsDNA (NEB), 1 mM DTT, and 2 µM concentrations of the proteins (RecA obtained from NEB M0249S, PfRad51, or PfRad54) to be tested for functional activity. .. The reaction mixture (135 µl) was preincubated at 37°C for 10 min, followed by addition of 15 µl of an initiation mixture (final concentration of 25 mM Tri-HCl, pH 7.5, 5% glycerol, 3 mM ATP, and 0.5 µM single-stranded binding protein [SSB from Stratagene or PfRPA1L or PfRPA1S]).

    Activity Assay:

    Article Title: Opposing Roles for Two Molecular Forms of Replication Protein A in Rad51-Rad54-Mediated DNA Recombination in Plasmodium falciparum
    Article Snippet: .. The assay mixture contained reaction buffer (25 mM Tris-HCl, pH 7.5, and 5% glycerol), 10 mM MgCl2 , 5 µM circular ΦX 174 RF I dsDNA (New England Biolabs [NEB]), 15 µM ΦX virion dsDNA (NEB), 1 mM DTT, and 2 µM concentrations of the proteins (RecA obtained from NEB M0249S, PfRad51, or PfRad54) to be tested for functional activity. .. The reaction mixture (135 µl) was preincubated at 37°C for 10 min, followed by addition of 15 µl of an initiation mixture (final concentration of 25 mM Tri-HCl, pH 7.5, 5% glycerol, 3 mM ATP, and 0.5 µM single-stranded binding protein [SSB from Stratagene or PfRPA1L or PfRPA1S]).

    Concentration Assay:

    Article Title: RecA homology search is promoted by mechanical stress along the scanned duplex DNA
    Article Snippet: These molecules were tethered to capillary walls at one end and to superparamagnetic beads (4.5 µm in diameter, 4 × 108 beads/ml, Invitrogen) at the other end. .. A 0.2 µl aliquot of each dsDNA at 7 µg/µl in RecA buffer (70 mM Tris–HCl, 10 mM MgCl2 and 5 mM dithiothreitol, pH 7.6) containing 1 mM ATP (or ATPγS) was mixed with free RecA (New England Biolabs) (final concentration 1 µM) or with preparations of RecA–ssDNA filaments (ranging in size from 57 to 7200 nt and final concentration 60 nM) and 1 µl of the beads. ..

    Article Title: The positioning of Chi sites allows the RecBCD pathway to suppress some genomic rearrangements
    Article Snippet: .. The filaments were initially prepared by incubating 0.06 μM ssDNA (final concentration ∼6 μM in bases) with 2 μM RecA (NEB) in the presence of 1 mM cofactor (ATP or dATP), 10 U/ml of pyruvate kinase, 3 mM phosphoenolpyruvate, and 0.2 μM single-stranded binding protein (SSB) (Epicentre) in RecA buffer (70 mM Tris–HCl, 10 mM MgCl2 and 5 mM dithiothreitol, pH 7.6) at 37°C for 10 min. FRET experiments followed the emission of the fluorescein label by using 493-nm excitation during 30 min; the emission was read as counts per second (cps) at 518 nm every one second. ..

    Labeling:

    Article Title: The poor homology stringency in the heteroduplex allows strand exchange to incorporate desirable mismatches without sacrificing recognition in vivo
    Article Snippet: B-form dsDNA obtained by ssDNA annealing Having evaluated the mismatch-dependent instability provided by the binding of free ssDNA to ssDNA bound to site I, we wanted to compare those results to the mismatch-dependent instability provided by the annealing of free ssDNA to form B-form dsDNA under the same conditions. .. Thus, we hybridized the same fluorescein and rhodamine labeled ssDNA molecules in the absence of RecA protein, as illustrated in Figure . ..

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    New England Biolabs reca protein
    <t>ssDNA-RecA+dsDNA</t> assay: force versus extension curves. ( A ) and were thus neglected; +, black: ssDNA before recombination (recorded in Binding Buffer); The best-fit parameters obtained using the worm like chain model are: l =5.4 μm and ξ=6.2 nm; × , blue: same molecule after extensive RecA polymerization (recorded in Binding Buffer+2 mM Mg 2+ to ensure good nucleoprotein filament stability). Best-fit parameters: l =6.9 μm and ξ=449.2 nm; ○, red: same molecule after recombination with 14 kb homologous dsDNA and rinsing to get rid of RecA (in Binding Buffer); □, green: 14 kb dsDNA used as a reference (in Binding buffer); best-fit parameters: l =4.7 μm and ξ=56.7 nm. In this example, the reaction product curve (○) was fitted with an ‘hybrid' curve consisting of 30% single-stranded <t>DNA</t> and 70% double-stranded DNA. Both increasing and decreasing force scans data are plotted, and were fitted independently with the model, leading to the two plain lines. They are almost superposed, showing the very weak hysteresis and good reproducibility of the experiments. 71±1%. ( B ) Histogram of the fraction of dsDNA in the hybrid molecule formed in presence of ATP, relative to the homology between the probe molecule and the substrate. Blue: experiments with 14 kb homologous DNA; red: experiments with 3.5 kb homologous DNA; green: experiments with sonicated 14 kb homologous DNA. ( C ) Histogram of the fraction of dsDNA in the hybrid molecule formed in the presence of ATPγS, relative to the homology between the probe molecule and the substrate. Same color meaning as in (B).
    Reca Protein, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ssDNA-RecA+dsDNA assay: force versus extension curves. ( A ) and were thus neglected; +, black: ssDNA before recombination (recorded in Binding Buffer); The best-fit parameters obtained using the worm like chain model are: l =5.4 μm and ξ=6.2 nm; × , blue: same molecule after extensive RecA polymerization (recorded in Binding Buffer+2 mM Mg 2+ to ensure good nucleoprotein filament stability). Best-fit parameters: l =6.9 μm and ξ=449.2 nm; ○, red: same molecule after recombination with 14 kb homologous dsDNA and rinsing to get rid of RecA (in Binding Buffer); □, green: 14 kb dsDNA used as a reference (in Binding buffer); best-fit parameters: l =4.7 μm and ξ=56.7 nm. In this example, the reaction product curve (○) was fitted with an ‘hybrid' curve consisting of 30% single-stranded DNA and 70% double-stranded DNA. Both increasing and decreasing force scans data are plotted, and were fitted independently with the model, leading to the two plain lines. They are almost superposed, showing the very weak hysteresis and good reproducibility of the experiments. 71±1%. ( B ) Histogram of the fraction of dsDNA in the hybrid molecule formed in presence of ATP, relative to the homology between the probe molecule and the substrate. Blue: experiments with 14 kb homologous DNA; red: experiments with 3.5 kb homologous DNA; green: experiments with sonicated 14 kb homologous DNA. ( C ) Histogram of the fraction of dsDNA in the hybrid molecule formed in the presence of ATPγS, relative to the homology between the probe molecule and the substrate. Same color meaning as in (B).

    Journal: The EMBO Journal

    Article Title: Mechanism of RecA-mediated homologous recombination revisited by single molecule nanomanipulation

    doi: 10.1038/sj.emboj.7601260

    Figure Lengend Snippet: ssDNA-RecA+dsDNA assay: force versus extension curves. ( A ) and were thus neglected; +, black: ssDNA before recombination (recorded in Binding Buffer); The best-fit parameters obtained using the worm like chain model are: l =5.4 μm and ξ=6.2 nm; × , blue: same molecule after extensive RecA polymerization (recorded in Binding Buffer+2 mM Mg 2+ to ensure good nucleoprotein filament stability). Best-fit parameters: l =6.9 μm and ξ=449.2 nm; ○, red: same molecule after recombination with 14 kb homologous dsDNA and rinsing to get rid of RecA (in Binding Buffer); □, green: 14 kb dsDNA used as a reference (in Binding buffer); best-fit parameters: l =4.7 μm and ξ=56.7 nm. In this example, the reaction product curve (○) was fitted with an ‘hybrid' curve consisting of 30% single-stranded DNA and 70% double-stranded DNA. Both increasing and decreasing force scans data are plotted, and were fitted independently with the model, leading to the two plain lines. They are almost superposed, showing the very weak hysteresis and good reproducibility of the experiments. 71±1%. ( B ) Histogram of the fraction of dsDNA in the hybrid molecule formed in presence of ATP, relative to the homology between the probe molecule and the substrate. Blue: experiments with 14 kb homologous DNA; red: experiments with 3.5 kb homologous DNA; green: experiments with sonicated 14 kb homologous DNA. ( C ) Histogram of the fraction of dsDNA in the hybrid molecule formed in the presence of ATPγS, relative to the homology between the probe molecule and the substrate. Same color meaning as in (B).

    Article Snippet: Annu Rev Biochem 63 : 991–1043 [ ] Kowalczykowski SC, Krupp R (1995) DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions .

    Techniques: dsDNA Assay, Binding Assay, Sonication

    dsDNA+ssDNA-RecA assay. Extension versus supercoiling curves. The measures are carried out in Binding Buffer, under 0.3 pN force. ( A ) Blue, dsDNA profile before incubation with nucleoprotein filaments; red, same molecule after 30 min incubation with 3.5 kb homologous preformed nucleoprotein filaments. The red plot is followed reversibly upon series of increasing and decreasing scans (increasing and decreasing σ, respectively). ( B ) Plot obtained after incubation with the same ssDNA, in the presence of SSB and in the absence of RecA. Black: naked DNA; red: first increasing scan from −420 to +100 turns. Green: ‘return' scan from +100 to −420 turns; After the first return scan, the green curve, identical to that of naked dsDNA, is reversibly followed during both increasing and decreasing scans. ( C ) Plots obtained after incubation with nucleoprotein filament assembled in the presence of ATPγS. Each color represents a different sequence of one increasing scan (plots with symbols) and one decreasing (full line without symbols) scan. This series of curves displays a stable hysteresis loop.

    Journal: The EMBO Journal

    Article Title: Mechanism of RecA-mediated homologous recombination revisited by single molecule nanomanipulation

    doi: 10.1038/sj.emboj.7601260

    Figure Lengend Snippet: dsDNA+ssDNA-RecA assay. Extension versus supercoiling curves. The measures are carried out in Binding Buffer, under 0.3 pN force. ( A ) Blue, dsDNA profile before incubation with nucleoprotein filaments; red, same molecule after 30 min incubation with 3.5 kb homologous preformed nucleoprotein filaments. The red plot is followed reversibly upon series of increasing and decreasing scans (increasing and decreasing σ, respectively). ( B ) Plot obtained after incubation with the same ssDNA, in the presence of SSB and in the absence of RecA. Black: naked DNA; red: first increasing scan from −420 to +100 turns. Green: ‘return' scan from +100 to −420 turns; After the first return scan, the green curve, identical to that of naked dsDNA, is reversibly followed during both increasing and decreasing scans. ( C ) Plots obtained after incubation with nucleoprotein filament assembled in the presence of ATPγS. Each color represents a different sequence of one increasing scan (plots with symbols) and one decreasing (full line without symbols) scan. This series of curves displays a stable hysteresis loop.

    Article Snippet: Annu Rev Biochem 63 : 991–1043 [ ] Kowalczykowski SC, Krupp R (1995) DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions .

    Techniques: Binding Assay, Incubation, Sequencing

    Kinetics of the dsDNA+ssDNA-RecA in solution assay. A 14 kb dsDNA is held at a stretching force of 2.3 pN, and 3.5 kb preformed homologous nucleoprotein filaments are injected at the beginning of recording. N is the number of turns applied on the dsDNA using the tweezers. Purple: applying negative supercoiling progressively by 100 turns steps every 600 s, in order to compensate for the untwisting action of the double strand invasion by the nucleofilament (see text). The degree of supercoiling corresponding to different sections of the curve are indicated in the figure; green: same conditions, except for imposing 400 turns negative supercoiling in one step at the beginning of incubation; red: same as green, except that the nucleoprotein filaments were assembled in the presence of ATP-γ-S instead of ATP; blue: control: same conditions as for the green curve, in the absence of nucleoprotein filament. Gray: control: same conditions as for the green curve, in the presence of heterologous nucleoprotein filament.

    Journal: The EMBO Journal

    Article Title: Mechanism of RecA-mediated homologous recombination revisited by single molecule nanomanipulation

    doi: 10.1038/sj.emboj.7601260

    Figure Lengend Snippet: Kinetics of the dsDNA+ssDNA-RecA in solution assay. A 14 kb dsDNA is held at a stretching force of 2.3 pN, and 3.5 kb preformed homologous nucleoprotein filaments are injected at the beginning of recording. N is the number of turns applied on the dsDNA using the tweezers. Purple: applying negative supercoiling progressively by 100 turns steps every 600 s, in order to compensate for the untwisting action of the double strand invasion by the nucleofilament (see text). The degree of supercoiling corresponding to different sections of the curve are indicated in the figure; green: same conditions, except for imposing 400 turns negative supercoiling in one step at the beginning of incubation; red: same as green, except that the nucleoprotein filaments were assembled in the presence of ATP-γ-S instead of ATP; blue: control: same conditions as for the green curve, in the absence of nucleoprotein filament. Gray: control: same conditions as for the green curve, in the presence of heterologous nucleoprotein filament.

    Article Snippet: Annu Rev Biochem 63 : 991–1043 [ ] Kowalczykowski SC, Krupp R (1995) DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions .

    Techniques: Injection, Incubation

    , for the passage of heterologies: Upon arrest of synapsis progression by the heterology, the synapsis rotation should be reversed by the release of torsional stress stored on DNA, induced by continuing RecA depolymerization no more compensated by strand invasion. This results in an untwisting of the heterologous zone if the invading and invaded DNAs are topologically linked downstream (not represented).

    Journal: The EMBO Journal

    Article Title: Mechanism of RecA-mediated homologous recombination revisited by single molecule nanomanipulation

    doi: 10.1038/sj.emboj.7601260

    Figure Lengend Snippet: , for the passage of heterologies: Upon arrest of synapsis progression by the heterology, the synapsis rotation should be reversed by the release of torsional stress stored on DNA, induced by continuing RecA depolymerization no more compensated by strand invasion. This results in an untwisting of the heterologous zone if the invading and invaded DNAs are topologically linked downstream (not represented).

    Article Snippet: Annu Rev Biochem 63 : 991–1043 [ ] Kowalczykowski SC, Krupp R (1995) DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions .

    Techniques:

    Highly conserved fingers domain residue is important for DinB's activity during strand exchange. A, schematic shows experimental setup for RecA-dependent strand-exchange experiments. Fluorescently-labeled dsDNA ( left ) is mixed with each of a set of ssDNA RecA filaments ( right ). Each in the set of filaments contains a different length of homology ( N ) to the labeled dsDNA (signified by gray, green, magenta, and blue lines ). In the dsDNA, the template strand is labeled with rhodamine ( yellow circle ), and the displaced strand is labeled with fluorescein ( white star ). These fluorophores are located 5 bp away from area of homology between dsDNA and ssDNA RecA filament (Δ L = 5 bp). The proximity of the rhodamine quenches fluorescein fluorescence until template and displaced strands are separated. Fluorescence increases when the ssDNA RecA filament invades the dsDNA, and DinB synthesizes DNA using the ssDNA filament as a primer. Five nucleotide insertions are needed to separate the displaced strand at the location of the fluorescent labels and relieve quenching. B, when only RecA or only DinB is mixed with the highest homology ssDNA RecA filament ( N = 75, blue filament in A ), the fluorescent labels are not efficiently separated, indicating that the dsDNA is still annealed at the location of the labels. When both proteins are present in the absence of dCTP, dGTP, and dTTP (dATP is present for nucleoprotein filament assembly), baseline fluorescence is observed. A RecA filament with full homology to the dsDNA ( N = 90) is used to determine maximum possible fluorescence in the assay. C, DinB stabilizes strand exchange in a homology-dependent manner. As homology increases between the ssDNA RecA filament and the fluorescently-labeled dsDNA, DinB efficiently separates the dsDNA at the location of the fluorescent labels. This indicates that increased homology allows DinB to more efficiently stabilize strand-exchange products. D, DinB(C66A) stabilizes strand-exchange products, but it does so with less efficiency than the native enzyme. N indicates the length of homology between dsDNA and ssDNA filament; Δ L indicates the distance between region of homology on dsDNA and fluorescent label; D init indicates the distance between fluorescent label and closet end of dsDNA; Δ F indicates the change in fluorescence measured in counts/s with respect to the fluorescence at 0 s. Experiments were performed in triplicate with similar results. Representative data are shown.

    Journal: The Journal of Biological Chemistry

    Article Title: Residues in the fingers domain of the translesion DNA polymerase DinB enable its unique participation in error-prone double-strand break repair

    doi: 10.1074/jbc.RA118.006233

    Figure Lengend Snippet: Highly conserved fingers domain residue is important for DinB's activity during strand exchange. A, schematic shows experimental setup for RecA-dependent strand-exchange experiments. Fluorescently-labeled dsDNA ( left ) is mixed with each of a set of ssDNA RecA filaments ( right ). Each in the set of filaments contains a different length of homology ( N ) to the labeled dsDNA (signified by gray, green, magenta, and blue lines ). In the dsDNA, the template strand is labeled with rhodamine ( yellow circle ), and the displaced strand is labeled with fluorescein ( white star ). These fluorophores are located 5 bp away from area of homology between dsDNA and ssDNA RecA filament (Δ L = 5 bp). The proximity of the rhodamine quenches fluorescein fluorescence until template and displaced strands are separated. Fluorescence increases when the ssDNA RecA filament invades the dsDNA, and DinB synthesizes DNA using the ssDNA filament as a primer. Five nucleotide insertions are needed to separate the displaced strand at the location of the fluorescent labels and relieve quenching. B, when only RecA or only DinB is mixed with the highest homology ssDNA RecA filament ( N = 75, blue filament in A ), the fluorescent labels are not efficiently separated, indicating that the dsDNA is still annealed at the location of the labels. When both proteins are present in the absence of dCTP, dGTP, and dTTP (dATP is present for nucleoprotein filament assembly), baseline fluorescence is observed. A RecA filament with full homology to the dsDNA ( N = 90) is used to determine maximum possible fluorescence in the assay. C, DinB stabilizes strand exchange in a homology-dependent manner. As homology increases between the ssDNA RecA filament and the fluorescently-labeled dsDNA, DinB efficiently separates the dsDNA at the location of the fluorescent labels. This indicates that increased homology allows DinB to more efficiently stabilize strand-exchange products. D, DinB(C66A) stabilizes strand-exchange products, but it does so with less efficiency than the native enzyme. N indicates the length of homology between dsDNA and ssDNA filament; Δ L indicates the distance between region of homology on dsDNA and fluorescent label; D init indicates the distance between fluorescent label and closet end of dsDNA; Δ F indicates the change in fluorescence measured in counts/s with respect to the fluorescence at 0 s. Experiments were performed in triplicate with similar results. Representative data are shown.

    Article Snippet: RecA nucleoprotein filament was formed by adding ssDNA (0.06 μ m ; and ), RecA (2 μ m ; New England Biolabs, Ipswich, MA), dATP (1 m m ), pyruvate kinase (10 units/ml), phosphoenolpyruvate (3 m m ), and single-stranded binding protein (0.2 μ m , Epicenter, Madison, WI) in RecA buffer (70 m m Tris-HCl, 10 m m MgCl2 , and 5 m m DTT, pH 7.6) at 37 °C for 10 min.

    Techniques: Activity Assay, Labeling, Fluorescence

    DinB(C66A) variant is deficient in RecA-independent strand displacement. A, graphic depiction of the DNA substrate used in these experiments. A 29-nt ssDNA primer ( gray line ) was annealed to a 90-nt template ( bottom black line ) as well as to a 75-nt fluorescently-labeled oligonucleotide ( top black line with the fluorophore represented by the star ). The 75-nt oligonucleotide displaced strand is composed of a 61-nt complementary to the 90-bp template and of a 14-nt unannealed flap located at the 5′ end. The fluorescein label (depicted by the star ) on the displaced strand was located on the first nucleotide of the 75-bp complementary region. If displaced and template strands are separated, fluorescence is altered. DinB must insert a single nucleotide onto the end of the primer to displace the labeled nucleotide. B, experiments with all dNTPs added show that DinB stabilizes strand displacement after a short lag (highlighted by the enlargement in C ) with greater efficiency than the DinB(C66A) variant. Initial velocities for both proteins from 0 to 50 s are not significantly different from zero. The rate of the DinB reaction from 100 to 200 s (46.40 ± 1.188 cps) is significantly higher than the velocity of the DinB(C66A) reaction at the same time point (11.80 ± 1.140 cps; p value

    Journal: The Journal of Biological Chemistry

    Article Title: Residues in the fingers domain of the translesion DNA polymerase DinB enable its unique participation in error-prone double-strand break repair

    doi: 10.1074/jbc.RA118.006233

    Figure Lengend Snippet: DinB(C66A) variant is deficient in RecA-independent strand displacement. A, graphic depiction of the DNA substrate used in these experiments. A 29-nt ssDNA primer ( gray line ) was annealed to a 90-nt template ( bottom black line ) as well as to a 75-nt fluorescently-labeled oligonucleotide ( top black line with the fluorophore represented by the star ). The 75-nt oligonucleotide displaced strand is composed of a 61-nt complementary to the 90-bp template and of a 14-nt unannealed flap located at the 5′ end. The fluorescein label (depicted by the star ) on the displaced strand was located on the first nucleotide of the 75-bp complementary region. If displaced and template strands are separated, fluorescence is altered. DinB must insert a single nucleotide onto the end of the primer to displace the labeled nucleotide. B, experiments with all dNTPs added show that DinB stabilizes strand displacement after a short lag (highlighted by the enlargement in C ) with greater efficiency than the DinB(C66A) variant. Initial velocities for both proteins from 0 to 50 s are not significantly different from zero. The rate of the DinB reaction from 100 to 200 s (46.40 ± 1.188 cps) is significantly higher than the velocity of the DinB(C66A) reaction at the same time point (11.80 ± 1.140 cps; p value

    Article Snippet: RecA nucleoprotein filament was formed by adding ssDNA (0.06 μ m ; and ), RecA (2 μ m ; New England Biolabs, Ipswich, MA), dATP (1 m m ), pyruvate kinase (10 units/ml), phosphoenolpyruvate (3 m m ), and single-stranded binding protein (0.2 μ m , Epicenter, Madison, WI) in RecA buffer (70 m m Tris-HCl, 10 m m MgCl2 , and 5 m m DTT, pH 7.6) at 37 °C for 10 min.

    Techniques: Variant Assay, Labeling, Fluorescence

    Experiments on 20 nt sequences using the emission of the donor in a FRET pair to study the homology sensitivity of ssDNA binding to the presynaptic filament, free ssDNA annealing and strand exchange. ( A ) Schematic of the binding of free ssDNA with the initiating strand in the presynaptic filament bringing fluorophores in close proximity. The large gray circles indicate RecA molecules. The yellow regions indicate Watson–Crick pairing. The red circle and the star correspond, respectively, to the positions of the rhodamine (acceptor) and fluorescein (donor) FRET partners. The star is green when the fluorescein is emitting and black when it is quenched by the nearby rhodamine. The magenta asterisks indicate the position of the mismatches for a partially matched case. ( B ) Emission of the fluorescein donor versus time for the perfectly matched (blue), mis4 (magenta), 3i (green), 1+2 (brown), pcDNA3 (solid black) and het (dashed black). ( C ) Annealing of free labeled ssDNA molecules without RecA protein. ( D ) Same as (B), except it is for the case shown schematically in panel (C). ( E ) Schematic of the strand exchange experiment. The outgoing, initiating and complementary strands are labeled o, i and c, respectively. ( F ) Fluorescence intensity as a function of time for the strand exchange experiment illustrated in panel (E) and for the same sequences shown in panel (B).

    Journal: Nucleic Acids Research

    Article Title: The poor homology stringency in the heteroduplex allows strand exchange to incorporate desirable mismatches without sacrificing recognition in vivo

    doi: 10.1093/nar/gkv610

    Figure Lengend Snippet: Experiments on 20 nt sequences using the emission of the donor in a FRET pair to study the homology sensitivity of ssDNA binding to the presynaptic filament, free ssDNA annealing and strand exchange. ( A ) Schematic of the binding of free ssDNA with the initiating strand in the presynaptic filament bringing fluorophores in close proximity. The large gray circles indicate RecA molecules. The yellow regions indicate Watson–Crick pairing. The red circle and the star correspond, respectively, to the positions of the rhodamine (acceptor) and fluorescein (donor) FRET partners. The star is green when the fluorescein is emitting and black when it is quenched by the nearby rhodamine. The magenta asterisks indicate the position of the mismatches for a partially matched case. ( B ) Emission of the fluorescein donor versus time for the perfectly matched (blue), mis4 (magenta), 3i (green), 1+2 (brown), pcDNA3 (solid black) and het (dashed black). ( C ) Annealing of free labeled ssDNA molecules without RecA protein. ( D ) Same as (B), except it is for the case shown schematically in panel (C). ( E ) Schematic of the strand exchange experiment. The outgoing, initiating and complementary strands are labeled o, i and c, respectively. ( F ) Fluorescence intensity as a function of time for the strand exchange experiment illustrated in panel (E) and for the same sequences shown in panel (B).

    Article Snippet: Thus, we hybridized the same fluorescein and rhodamine labeled ssDNA molecules in the absence of RecA protein, as illustrated in Figure .

    Techniques: Binding Assay, Labeling, Fluorescence

    (A) Role of PfRPA1L and PfRPA1S during SSE activity of PfRad51. (I) PfRad51 and SSB. (II) PfRad51 and PfRPA1L. (III) PfRad51 and PfRPA1S. (B) PfRPA1S downregulates the function of PfRPA1L. (I) PfRad51, 0.5 μM PfRPA1L, and 0.5 μM PfRPA1S. (II) PfRad51, 0.5 μM PfRPA1L, and 0.75 μM PfRPA1S. (III) PfRad51, 0.5 μM PfRPA1L, and 1.0 μM PfRPA1S. (IV) PfRad51, 0.5 μM PfRPA1L, and 2.0 μM PfRPA1S. (V) PfRad51 and 0.5 μM PfRPA1S preincubated for 10 min, followed by addition of 0.5 μM PfRPA1L. (VI) PfRad51 and 0.5 μM PfRPA1S preincubated for 10 min, followed by addition of 1.0 μM of PfRPA1L. (C) Role of PfRad51, PfRPA1L, and PfRPA1S in the presence of the bacterial homologue RecA and SSB. (I) PfRad51, 0.5 μM SSB, and 0.5 μM PfRPA1S. (II) RecA and PfRPA1L. (III) RecA and PfRPA1S. (IV) RecA, 0.5 μM PfRPA1L, and 0.5 μM PfRPA1S. Aliquots were collected at time points (min) indicated above each lane and quenched with stop solution, and products were revealed on 1% TAE agarose gel, followed by EtBr staining. Lds, linear double-stranded DNA; NC, nicked circular dsDNA; JM, joint molecule. These figures are a representative assay of three biologically independent strand exchange assays.

    Journal: mBio

    Article Title: Opposing Roles for Two Molecular Forms of Replication Protein A in Rad51-Rad54-Mediated DNA Recombination in Plasmodium falciparum

    doi: 10.1128/mBio.00252-13

    Figure Lengend Snippet: (A) Role of PfRPA1L and PfRPA1S during SSE activity of PfRad51. (I) PfRad51 and SSB. (II) PfRad51 and PfRPA1L. (III) PfRad51 and PfRPA1S. (B) PfRPA1S downregulates the function of PfRPA1L. (I) PfRad51, 0.5 μM PfRPA1L, and 0.5 μM PfRPA1S. (II) PfRad51, 0.5 μM PfRPA1L, and 0.75 μM PfRPA1S. (III) PfRad51, 0.5 μM PfRPA1L, and 1.0 μM PfRPA1S. (IV) PfRad51, 0.5 μM PfRPA1L, and 2.0 μM PfRPA1S. (V) PfRad51 and 0.5 μM PfRPA1S preincubated for 10 min, followed by addition of 0.5 μM PfRPA1L. (VI) PfRad51 and 0.5 μM PfRPA1S preincubated for 10 min, followed by addition of 1.0 μM of PfRPA1L. (C) Role of PfRad51, PfRPA1L, and PfRPA1S in the presence of the bacterial homologue RecA and SSB. (I) PfRad51, 0.5 μM SSB, and 0.5 μM PfRPA1S. (II) RecA and PfRPA1L. (III) RecA and PfRPA1S. (IV) RecA, 0.5 μM PfRPA1L, and 0.5 μM PfRPA1S. Aliquots were collected at time points (min) indicated above each lane and quenched with stop solution, and products were revealed on 1% TAE agarose gel, followed by EtBr staining. Lds, linear double-stranded DNA; NC, nicked circular dsDNA; JM, joint molecule. These figures are a representative assay of three biologically independent strand exchange assays.

    Article Snippet: The assay mixture contained reaction buffer (25 mM Tris-HCl, pH 7.5, and 5% glycerol), 10 mM MgCl2 , 5 µM circular ΦX 174 RF I dsDNA (New England Biolabs [NEB]), 15 µM ΦX virion dsDNA (NEB), 1 mM DTT, and 2 µM concentrations of the proteins (RecA obtained from NEB M0249S, PfRad51, or PfRad54) to be tested for functional activity.

    Techniques: Activity Assay, Agarose Gel Electrophoresis, Staining