single stranded dna binding protein ssb Search Results


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  • 97
    New England Biolabs et ssb
    Et Ssb, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/et ssb/product/New England Biolabs
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    99
    Thermo Fisher single stranded dna
    EV proteins and <t>RNA</t> are necessary to increase survival of cryptococci inside macrophages. a IPRs of ICB180 growing alone (ICB180) and in the presence of 10 μg of EVs isolated from acapsular strain R265ΔCap10 (EVs R265ΔCap10 ) or heat-inactivated EVs R265ΔCap10 (EVs R265ΔCap10 hk ) added at different stages of infection: during yeast opsonisation using PHS (opsonisation), J774 activation (activation) or during incubation with both macrophages and ICB180 yeast cells (infection; see also Fig. 3a ). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 8 to 9 independent experiments with 147–301 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P = 0.0117), significant difference; and ns ( P > 0.05), not significantly different. b Schematic drawing of the EV and treatments performed towards protein degradation via proteinase K, lipids degradation via sodium deoxycholate, double-stranded <t>DNA</t> (dsDNA) degradation via dsDNase, single-stranded DNA (ssDNA) and single-stranded regions of RNA degradation via S1 nuclease and further RNA degradation, including RNA duplexes, via RNase cocktail of RNase A and T1. c IPR values of ICB180 are increased in the presence of 10 μg of EVs (or 50 μg—symbols with thicker borders) isolated from R265 (+EVs R265 ), EVs R265 treated with S1 nuclease (+EVs R265 S1 nuclease) and EVs R265 treated with dsDNase (+EVs R265 dsDNase) but not when EVs treated with proteinase K (+EVs R265 proteinase K), sodium deoxycholate (+EVs R265 detergent) or RNase cocktail (+EVs R265 RNases) were used. Data are representative of results from 10 to 15 independent experiments with 1181–2691 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P ≤ 0.05), significant difference; ** ( P ≤ 0.01), significant difference, *** ( P ≤ 0.001), significant difference and ns ( P > 0.05), not significantly different
    Single Stranded Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1265 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore single stranded dna binding protein ssb
    EV proteins and <t>RNA</t> are necessary to increase survival of cryptococci inside macrophages. a IPRs of ICB180 growing alone (ICB180) and in the presence of 10 μg of EVs isolated from acapsular strain R265ΔCap10 (EVs R265ΔCap10 ) or heat-inactivated EVs R265ΔCap10 (EVs R265ΔCap10 hk ) added at different stages of infection: during yeast opsonisation using PHS (opsonisation), J774 activation (activation) or during incubation with both macrophages and ICB180 yeast cells (infection; see also Fig. 3a ). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 8 to 9 independent experiments with 147–301 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P = 0.0117), significant difference; and ns ( P > 0.05), not significantly different. b Schematic drawing of the EV and treatments performed towards protein degradation via proteinase K, lipids degradation via sodium deoxycholate, double-stranded <t>DNA</t> (dsDNA) degradation via dsDNase, single-stranded DNA (ssDNA) and single-stranded regions of RNA degradation via S1 nuclease and further RNA degradation, including RNA duplexes, via RNase cocktail of RNase A and T1. c IPR values of ICB180 are increased in the presence of 10 μg of EVs (or 50 μg—symbols with thicker borders) isolated from R265 (+EVs R265 ), EVs R265 treated with S1 nuclease (+EVs R265 S1 nuclease) and EVs R265 treated with dsDNase (+EVs R265 dsDNase) but not when EVs treated with proteinase K (+EVs R265 proteinase K), sodium deoxycholate (+EVs R265 detergent) or RNase cocktail (+EVs R265 RNases) were used. Data are representative of results from 10 to 15 independent experiments with 1181–2691 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P ≤ 0.05), significant difference; ** ( P ≤ 0.01), significant difference, *** ( P ≤ 0.001), significant difference and ns ( P > 0.05), not significantly different
    Single Stranded Dna Binding Protein Ssb, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega single stranded dna binding protein ssb
    EV proteins and <t>RNA</t> are necessary to increase survival of cryptococci inside macrophages. a IPRs of ICB180 growing alone (ICB180) and in the presence of 10 μg of EVs isolated from acapsular strain R265ΔCap10 (EVs R265ΔCap10 ) or heat-inactivated EVs R265ΔCap10 (EVs R265ΔCap10 hk ) added at different stages of infection: during yeast opsonisation using PHS (opsonisation), J774 activation (activation) or during incubation with both macrophages and ICB180 yeast cells (infection; see also Fig. 3a ). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 8 to 9 independent experiments with 147–301 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P = 0.0117), significant difference; and ns ( P > 0.05), not significantly different. b Schematic drawing of the EV and treatments performed towards protein degradation via proteinase K, lipids degradation via sodium deoxycholate, double-stranded <t>DNA</t> (dsDNA) degradation via dsDNase, single-stranded DNA (ssDNA) and single-stranded regions of RNA degradation via S1 nuclease and further RNA degradation, including RNA duplexes, via RNase cocktail of RNase A and T1. c IPR values of ICB180 are increased in the presence of 10 μg of EVs (or 50 μg—symbols with thicker borders) isolated from R265 (+EVs R265 ), EVs R265 treated with S1 nuclease (+EVs R265 S1 nuclease) and EVs R265 treated with dsDNase (+EVs R265 dsDNase) but not when EVs treated with proteinase K (+EVs R265 proteinase K), sodium deoxycholate (+EVs R265 detergent) or RNase cocktail (+EVs R265 RNases) were used. Data are representative of results from 10 to 15 independent experiments with 1181–2691 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P ≤ 0.05), significant difference; ** ( P ≤ 0.01), significant difference, *** ( P ≤ 0.001), significant difference and ns ( P > 0.05), not significantly different
    Single Stranded Dna Binding Protein Ssb, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Stratagene single stranded dna binding protein ssb
    EV proteins and <t>RNA</t> are necessary to increase survival of cryptococci inside macrophages. a IPRs of ICB180 growing alone (ICB180) and in the presence of 10 μg of EVs isolated from acapsular strain R265ΔCap10 (EVs R265ΔCap10 ) or heat-inactivated EVs R265ΔCap10 (EVs R265ΔCap10 hk ) added at different stages of infection: during yeast opsonisation using PHS (opsonisation), J774 activation (activation) or during incubation with both macrophages and ICB180 yeast cells (infection; see also Fig. 3a ). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 8 to 9 independent experiments with 147–301 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P = 0.0117), significant difference; and ns ( P > 0.05), not significantly different. b Schematic drawing of the EV and treatments performed towards protein degradation via proteinase K, lipids degradation via sodium deoxycholate, double-stranded <t>DNA</t> (dsDNA) degradation via dsDNase, single-stranded DNA (ssDNA) and single-stranded regions of RNA degradation via S1 nuclease and further RNA degradation, including RNA duplexes, via RNase cocktail of RNase A and T1. c IPR values of ICB180 are increased in the presence of 10 μg of EVs (or 50 μg—symbols with thicker borders) isolated from R265 (+EVs R265 ), EVs R265 treated with S1 nuclease (+EVs R265 S1 nuclease) and EVs R265 treated with dsDNase (+EVs R265 dsDNase) but not when EVs treated with proteinase K (+EVs R265 proteinase K), sodium deoxycholate (+EVs R265 detergent) or RNase cocktail (+EVs R265 RNases) were used. Data are representative of results from 10 to 15 independent experiments with 1181–2691 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P ≤ 0.05), significant difference; ** ( P ≤ 0.01), significant difference, *** ( P ≤ 0.001), significant difference and ns ( P > 0.05), not significantly different
    Single Stranded Dna Binding Protein Ssb, supplied by Stratagene, used in various techniques. Bioz Stars score: 85/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare e coli single stranded dna binding protein ssb
    Enhanced binding by Rep proteins is specific and does not require helicase activity. (A) Effect of ssDBPs on binding of MBPRep78 to a linear <t>DNA</t> substrate containing the RRS. The radiolabeled RRS fragment (1,000 cpm) was incubated with MBPRep78 (the numbers at the top indicate the amounts of protein, in micrograms). The ssDBPs were incubated with the probe in the absence (−) or presence (+) of MBPRep78. Binding was enhanced by the HSV-1 ICP8 and RPA proteins but not by the E. coli <t>SSB</t> protein. A constant amount of Rep (indicated by the asterisk below) was assessed for the effect of the ssDBPs. The lane marked “RRS alone” does not contain any added protein. The positions of free DNA probe (F) and DNA probe bound in a Rep complex (B) are shown to the right. (B) Enhanced binding of Rep68 to the RRS fragment. Either 1 ng (+) or 2 ng (++) of His-tagged recombinant Rep68 protein (Rep68H) was incubated with a radiolabeled RRS fragment together with the indicated ssDBP. (C) ssDBPs increase binding of a mutant Rep protein that has lost helicase activity. A mutant Rep protein (Y121H/K340H) was incubated in an EMSA with a 32 P-labeled linear DNA fragment containing the RRS, in the presence or absence of the indicated ssDBPs. (D) Complex dissociation was assessed with Rep68H alone or in the presence of Ad-DBP. The Rep68H protein was bound to the RRS fragment and then dissociation was assessed by the addition of increasing amounts of unlabeled competitor DNA to assembled complexes. Binding assays were analyzed by gel electrophoresis and relative binding was quantitated by PhosphorImager analysis of gels.
    E Coli Single Stranded Dna Binding Protein Ssb, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam recombinant e coli single stranded dna binding ssb protein
    YB-1C also forms ssDNA nucleoprotein filaments, unwinds secondary structures and is compatible with mRNA translation. ( A ) AFM images of circular ssDNA (M13) complexed with indicated proteins at saturation. M13 ssDNA, 2 nM; <t>SSB,</t> 1 μM; YB-1, 0.5 μM; aa 1–219, 0.7 μM; aa 1–180 (YB-1C), 2 μM. Lower right panel: contour length of ssDNA or mRNA nucleoprotein filaments. N = 10. Blue bar: mean. ( B ) A <t>DNA</t> construct consisting of two circular ssDNA (1500 and 5000 nt) separated by a dsDNA stalk (300 bp) complexed with either YB-1 or YB-1C. ssDNA, 0.5 nM; YB-1, 0.5 μM; aa 1–180 (YB-1C), 2 μM. ( C ) Left panel: electrophoretic mobility of mRNA or ssDNA in the presence of different proteins. The presence of YB-1C at saturation decreases EtBr fluorescence of the mRNA band, as observed for ssDNA nucleoprotein filament with Escherichia coli SSB. Right panel: quantification of ethidium fluorescence of three replicates under the same condition. ( D ) In vitro translation assay in RRL. mRNA was pre-incubated with proteins for 10 min before addition in RRL for 10 min. Note the inhibition of mRNA translation by YB-1 but not by YB-1C. mRNA: 4 nM. Anti-YB-1 and anti-luciferase primary antibodies. Both endogenous and overexpressed YB-1 constructs are recognized by the anti-YB-1 antibody (see also Supplementary Figure S1 ).
    Recombinant E Coli Single Stranded Dna Binding Ssb Protein, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Stratagene recombinant e coli single strand dna binding protein ssb
    Ideication of the UV cross-linked amino acid residue in <t>SSB</t> by nanoelectrospray tandem mass spectrometry of the <t>peptide–DNA</t> heteroconjugate. ( A ) Nanoelectrospray mass spectrum obtained from the Oligo R3 eluate after tryptic digest, IMAC purification, and phosphodiesterase I digest. The major ion signal is attributable to a triply charged species at m/z 779.62. ( B ) First MS/MS fragmentation regime: product ion spectrum of the triply charged species at m/z 779.62 at a Q 0 setting of 40 V (about 60 eV collision energy in LRF). The major fragments corresponding to the fragmentation of a trinucleotide with the sequence TpGpX are labeled. The fragments corresponding to the loss of the former 5-iodouracil are marked with triangles. No peptide backbone cleavage products are observable. ( C ) Second MS/MS fragmentation regime: product ion spectrum of the same species at a Q 0 setting of 60 V (about 120 eV collision energy in LRF). The region above m/z 300 is enlarged 46-fold showing fragments deriving from the cleavages of the amide bond. The fragment ions y 8 and y 9 unambiguously demonstrate that W88 was cross-linked to the DNA. Sequence specific fragment ions are labeled. ( D ) in bold face; amino acid one-letter code underlined in bold face. The lower part shows the fragmentation of the peptide at a collision energy of about 120 eV. The peptide fragment ions are labeled according to Biemann (1988). ( E ) Product ion spectrum of the triply charged peptide–trinucleotide heteroconjugate at m/z 615.5, revealing Trp-54 to be the second UV-cross-linked amino acid residue.
    Recombinant E Coli Single Strand Dna Binding Protein Ssb, supplied by Stratagene, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore e coli ssb
    Ideication of the UV cross-linked amino acid residue in <t>SSB</t> by nanoelectrospray tandem mass spectrometry of the <t>peptide–DNA</t> heteroconjugate. ( A ) Nanoelectrospray mass spectrum obtained from the Oligo R3 eluate after tryptic digest, IMAC purification, and phosphodiesterase I digest. The major ion signal is attributable to a triply charged species at m/z 779.62. ( B ) First MS/MS fragmentation regime: product ion spectrum of the triply charged species at m/z 779.62 at a Q 0 setting of 40 V (about 60 eV collision energy in LRF). The major fragments corresponding to the fragmentation of a trinucleotide with the sequence TpGpX are labeled. The fragments corresponding to the loss of the former 5-iodouracil are marked with triangles. No peptide backbone cleavage products are observable. ( C ) Second MS/MS fragmentation regime: product ion spectrum of the same species at a Q 0 setting of 60 V (about 120 eV collision energy in LRF). The region above m/z 300 is enlarged 46-fold showing fragments deriving from the cleavages of the amide bond. The fragment ions y 8 and y 9 unambiguously demonstrate that W88 was cross-linked to the DNA. Sequence specific fragment ions are labeled. ( D ) in bold face; amino acid one-letter code underlined in bold face. The lower part shows the fragmentation of the peptide at a collision energy of about 120 eV. The peptide fragment ions are labeled according to Biemann (1988). ( E ) Product ion spectrum of the triply charged peptide–trinucleotide heteroconjugate at m/z 615.5, revealing Trp-54 to be the second UV-cross-linked amino acid residue.
    E Coli Ssb, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore coli single stranded dna binding protein ssb
    DdrC stimulates <t>DNA</t> annealing. Kinetics of two complementary 67-mer oligonucleotides annealing in the absence (w/o protein) or the presence of DdrC, T4 gp32 or SSB using a DAPI fluorescence-based method. The 67-mer oligonucleotide (200 nM) was mixed in 1 ml of reaction buffer with 0.2 μM DdrC protein, or 0.1 μM T4 gp32, or 0.1 μM SSB from E . coli prior to addition of the reverse oligonucleotide. The extent of DNA annealing is defined as follows: (observed fluorescence—67-mer <t>ssDNA</t> fluorescence) x 100 / 67-mer ds DNA fluorescence.
    Coli Single Stranded Dna Binding Protein Ssb, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    EV proteins and RNA are necessary to increase survival of cryptococci inside macrophages. a IPRs of ICB180 growing alone (ICB180) and in the presence of 10 μg of EVs isolated from acapsular strain R265ΔCap10 (EVs R265ΔCap10 ) or heat-inactivated EVs R265ΔCap10 (EVs R265ΔCap10 hk ) added at different stages of infection: during yeast opsonisation using PHS (opsonisation), J774 activation (activation) or during incubation with both macrophages and ICB180 yeast cells (infection; see also Fig. 3a ). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 8 to 9 independent experiments with 147–301 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P = 0.0117), significant difference; and ns ( P > 0.05), not significantly different. b Schematic drawing of the EV and treatments performed towards protein degradation via proteinase K, lipids degradation via sodium deoxycholate, double-stranded DNA (dsDNA) degradation via dsDNase, single-stranded DNA (ssDNA) and single-stranded regions of RNA degradation via S1 nuclease and further RNA degradation, including RNA duplexes, via RNase cocktail of RNase A and T1. c IPR values of ICB180 are increased in the presence of 10 μg of EVs (or 50 μg—symbols with thicker borders) isolated from R265 (+EVs R265 ), EVs R265 treated with S1 nuclease (+EVs R265 S1 nuclease) and EVs R265 treated with dsDNase (+EVs R265 dsDNase) but not when EVs treated with proteinase K (+EVs R265 proteinase K), sodium deoxycholate (+EVs R265 detergent) or RNase cocktail (+EVs R265 RNases) were used. Data are representative of results from 10 to 15 independent experiments with 1181–2691 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P ≤ 0.05), significant difference; ** ( P ≤ 0.01), significant difference, *** ( P ≤ 0.001), significant difference and ns ( P > 0.05), not significantly different

    Journal: Nature Communications

    Article Title: Pathogen-derived extracellular vesicles mediate virulence in the fatal human pathogen Cryptococcus gattii

    doi: 10.1038/s41467-018-03991-6

    Figure Lengend Snippet: EV proteins and RNA are necessary to increase survival of cryptococci inside macrophages. a IPRs of ICB180 growing alone (ICB180) and in the presence of 10 μg of EVs isolated from acapsular strain R265ΔCap10 (EVs R265ΔCap10 ) or heat-inactivated EVs R265ΔCap10 (EVs R265ΔCap10 hk ) added at different stages of infection: during yeast opsonisation using PHS (opsonisation), J774 activation (activation) or during incubation with both macrophages and ICB180 yeast cells (infection; see also Fig. 3a ). Data are presented as scattered dot plots with lines representing their medians. Data are representative of results from 8 to 9 independent experiments with 147–301 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P = 0.0117), significant difference; and ns ( P > 0.05), not significantly different. b Schematic drawing of the EV and treatments performed towards protein degradation via proteinase K, lipids degradation via sodium deoxycholate, double-stranded DNA (dsDNA) degradation via dsDNase, single-stranded DNA (ssDNA) and single-stranded regions of RNA degradation via S1 nuclease and further RNA degradation, including RNA duplexes, via RNase cocktail of RNase A and T1. c IPR values of ICB180 are increased in the presence of 10 μg of EVs (or 50 μg—symbols with thicker borders) isolated from R265 (+EVs R265 ), EVs R265 treated with S1 nuclease (+EVs R265 S1 nuclease) and EVs R265 treated with dsDNase (+EVs R265 dsDNase) but not when EVs treated with proteinase K (+EVs R265 proteinase K), sodium deoxycholate (+EVs R265 detergent) or RNase cocktail (+EVs R265 RNases) were used. Data are representative of results from 10 to 15 independent experiments with 1181–2691 total number of yeasts counted for each sample. Wilcoxon paired t test where * ( P ≤ 0.05), significant difference; ** ( P ≤ 0.01), significant difference, *** ( P ≤ 0.001), significant difference and ns ( P > 0.05), not significantly different

    Article Snippet: To degrade single-stranded DNA and RNA deprived of double-stranded regions, 0.4 μl of S1 nuclease (Thermo Fisher Scientific #EN0321) was added to 20 μl EVsR265 for 30 min at room temperature.

    Techniques: Isolation, Infection, Activation Assay, Incubation

    Detection of ssDNA gaps on ongoing replication forks. ( A ) Neutral comet assay with or without ssDNA-specific S1 endonuclease 24 h after 0 or 5 J/m 2 in XP-C cells depleted for TLS Pols with shRNA. Results are expressed as tail moment (average ± SEM) from ≥ 50 comets per condition of four independent experiments performed in duplicate. The significance of differences between UV-exposed cells compared to its respective untreated control, irradiated cells with or without S1, and between cell lines was assessed by one-way ANOVA followed by Bonferroni test. ( B ) Scheme of DNA fiber assay with the ssDNA-specific nuclease S1 for the detection of ssDNA gaps on ongoing forks. DNA fiber assay with or without S1 nuclease in XP-C cor ( C ), XP-C shCT and XP-C shRev3L cells ( D ) and XP-C shCT and XP-C shRev1 ( E ) upon 0 or 20 J/m 2 UVC and represented by CldU/IdU ratios (average ± SEM) from two independent experiments each (≥100 fibers for 0 J/m 2 and ≥150 fibers for 20 J/m 2 ). Statistical significances were determined by one-way ANOVA followed by Bonferroni test (ns, non significant; * P

    Journal: Nucleic Acids Research

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells

    doi: 10.1093/nar/gkw280

    Figure Lengend Snippet: Detection of ssDNA gaps on ongoing replication forks. ( A ) Neutral comet assay with or without ssDNA-specific S1 endonuclease 24 h after 0 or 5 J/m 2 in XP-C cells depleted for TLS Pols with shRNA. Results are expressed as tail moment (average ± SEM) from ≥ 50 comets per condition of four independent experiments performed in duplicate. The significance of differences between UV-exposed cells compared to its respective untreated control, irradiated cells with or without S1, and between cell lines was assessed by one-way ANOVA followed by Bonferroni test. ( B ) Scheme of DNA fiber assay with the ssDNA-specific nuclease S1 for the detection of ssDNA gaps on ongoing forks. DNA fiber assay with or without S1 nuclease in XP-C cor ( C ), XP-C shCT and XP-C shRev3L cells ( D ) and XP-C shCT and XP-C shRev1 ( E ) upon 0 or 20 J/m 2 UVC and represented by CldU/IdU ratios (average ± SEM) from two independent experiments each (≥100 fibers for 0 J/m 2 and ≥150 fibers for 20 J/m 2 ). Statistical significances were determined by one-way ANOVA followed by Bonferroni test (ns, non significant; * P

    Article Snippet: ssDNA detection by modified neutral comet assay The single-stranded DNA (ssDNA)-specific S1 endonuclease from Aspergillus oryzae (Invitrogen, Life Technologies) ( ) was used to generate double-strand breaks (DSB) from UV-induced ssDNA sites ( ).

    Techniques: Neutral Comet Assay, shRNA, Irradiation

    Effects of 6-4PP and CPD photorepair on ssDNA gaps formation and postreplication repair (PRR) tracts. XP-C shCT cells were transduced with Ad6-4phr, AdCPDphr or mock-treated. ( A ) Scheme of DNA fiber assay with the ssDNA-specific S1 nuclease in photorepair conditions. ( B ) CldU/IdU ratios from XP-C cells exposed to 0 or 50 J/m 2 and treated or not with S1 from three independent experiments (≥100 fibers for 0 J/m 2 and ≥150 fibers for 50 J/m 2 each). ( C ) Scheme for PRR tract detection in photorepair conditions. ( D ) Quantification of PRR tracts density 24 h upon exposure to 0 or 20 J/m 2 . Statistical significances were determined by one-way ANOVA followed by Bonferroni test (ns, non significant; * P

    Journal: Nucleic Acids Research

    Article Title: Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells

    doi: 10.1093/nar/gkw280

    Figure Lengend Snippet: Effects of 6-4PP and CPD photorepair on ssDNA gaps formation and postreplication repair (PRR) tracts. XP-C shCT cells were transduced with Ad6-4phr, AdCPDphr or mock-treated. ( A ) Scheme of DNA fiber assay with the ssDNA-specific S1 nuclease in photorepair conditions. ( B ) CldU/IdU ratios from XP-C cells exposed to 0 or 50 J/m 2 and treated or not with S1 from three independent experiments (≥100 fibers for 0 J/m 2 and ≥150 fibers for 50 J/m 2 each). ( C ) Scheme for PRR tract detection in photorepair conditions. ( D ) Quantification of PRR tracts density 24 h upon exposure to 0 or 20 J/m 2 . Statistical significances were determined by one-way ANOVA followed by Bonferroni test (ns, non significant; * P

    Article Snippet: ssDNA detection by modified neutral comet assay The single-stranded DNA (ssDNA)-specific S1 endonuclease from Aspergillus oryzae (Invitrogen, Life Technologies) ( ) was used to generate double-strand breaks (DSB) from UV-induced ssDNA sites ( ).

    Techniques: Transduction

    Activities of WRNp on an unmodified, 5′-overhang DNA substrate. ( A ) Assay of WRNp helicase activity on the 5′-overhang DNA substrate. The unmodified substrate was incubated with or without WRNp (150 fmol) for 1 h at 37°C and the DNA products were analyzed on a non-denaturing 12% polyacrylamide gel. Under these non-denaturing electrophoretic conditions, migration of double-stranded DNA species is dictated by the length of the unlabeled strand (data not shown) and thus WRNp exonuclease degradation products cannot be resolved by this method. As a marker for the migration of the labeled strand, the DNA substrate was loaded onto the gel immediately following heat denaturation (denoted by the triangle). The positions of the intact double-stranded substrate and the labeled single-stranded 32mer are denoted to the left. ( B ) Assay of exo III, Klenow and WRNp 3′→5′ exonuclease activities on the unmodified, 5′-overhang DNA substrate. The unmodified DNA substrate (depicted at the top) was incubated without enzyme (–enzyme) or with exo III (1 U), Klenow (2 U) or WRNp (120 fmol) at 37°C for 1 h. After heating at 90°C, the single-stranded DNA products were separated on a 15% denaturing polyacrylamide gel and visualized by phosphorimaging. The identities of the 3′-end nucleotide for each single-stranded degradation product are denoted to the right.

    Journal: Nucleic Acids Research

    Article Title: Selective blockage of the 3?- > 5? exonuclease activity of WRN protein by certain oxidative modifications and bulky lesions in DNA

    doi:

    Figure Lengend Snippet: Activities of WRNp on an unmodified, 5′-overhang DNA substrate. ( A ) Assay of WRNp helicase activity on the 5′-overhang DNA substrate. The unmodified substrate was incubated with or without WRNp (150 fmol) for 1 h at 37°C and the DNA products were analyzed on a non-denaturing 12% polyacrylamide gel. Under these non-denaturing electrophoretic conditions, migration of double-stranded DNA species is dictated by the length of the unlabeled strand (data not shown) and thus WRNp exonuclease degradation products cannot be resolved by this method. As a marker for the migration of the labeled strand, the DNA substrate was loaded onto the gel immediately following heat denaturation (denoted by the triangle). The positions of the intact double-stranded substrate and the labeled single-stranded 32mer are denoted to the left. ( B ) Assay of exo III, Klenow and WRNp 3′→5′ exonuclease activities on the unmodified, 5′-overhang DNA substrate. The unmodified DNA substrate (depicted at the top) was incubated without enzyme (–enzyme) or with exo III (1 U), Klenow (2 U) or WRNp (120 fmol) at 37°C for 1 h. After heating at 90°C, the single-stranded DNA products were separated on a 15% denaturing polyacrylamide gel and visualized by phosphorimaging. The identities of the 3′-end nucleotide for each single-stranded degradation product are denoted to the right.

    Article Snippet: Single-stranded DNA oligomers (21mer, 32mer and 43mer) containing only normal nucleotides were obtained from Gibco BRL.

    Techniques: Activity Assay, Incubation, Migration, Marker, Labeling

    Course of the in vitro selection. As a measure for the enrichment of biotinylated d -glucagon-binding DNA sequences, the ratio of the fraction of library bound to the target immobilized on streptavidin or neutravidin beads versus the applied biotinylated peptide ( d -glucagon) concentration is plotted against the selection round numbers. A steep increase in binding is visible in rounds 6 to 8 followed by stagnation in rounds 9 to 12. In the following rounds the regular alternation between mutagenic and standard PCR results in alternating ratios.

    Journal: The Journal of Biological Chemistry

    Article Title: A Mixed Mirror-image DNA/RNA Aptamer Inhibits Glucagon and Acutely Improves Glucose Tolerance in Models of Type 1 and Type 2 Diabetes *

    doi: 10.1074/jbc.M112.444414

    Figure Lengend Snippet: Course of the in vitro selection. As a measure for the enrichment of biotinylated d -glucagon-binding DNA sequences, the ratio of the fraction of library bound to the target immobilized on streptavidin or neutravidin beads versus the applied biotinylated peptide ( d -glucagon) concentration is plotted against the selection round numbers. A steep increase in binding is visible in rounds 6 to 8 followed by stagnation in rounds 9 to 12. In the following rounds the regular alternation between mutagenic and standard PCR results in alternating ratios.

    Article Snippet: In Vitro Selection DNA aptamers were selected by incubating the C terminally biotinylated d -glucagon with the single-stranded DNA library that had been 5′-labeled with [γ-32 P]ATP by T4 polynucleotide kinase (Invitrogen, Darmstadt, Germany) in selection buffer (20 mm Tris, pH 7.4, 150 mm NaCl, 5 mm KCl, 1 mm MgCl2, 1 mm CaCl2 , 0.1% Tween 20, 0.1% CHAPS, 100 μg/ml of human serum albumin, 10 μg/ml of yeast RNA).

    Techniques: In Vitro, Selection, Binding Assay, Concentration Assay, Polymerase Chain Reaction

    Enhanced binding by Rep proteins is specific and does not require helicase activity. (A) Effect of ssDBPs on binding of MBPRep78 to a linear DNA substrate containing the RRS. The radiolabeled RRS fragment (1,000 cpm) was incubated with MBPRep78 (the numbers at the top indicate the amounts of protein, in micrograms). The ssDBPs were incubated with the probe in the absence (−) or presence (+) of MBPRep78. Binding was enhanced by the HSV-1 ICP8 and RPA proteins but not by the E. coli SSB protein. A constant amount of Rep (indicated by the asterisk below) was assessed for the effect of the ssDBPs. The lane marked “RRS alone” does not contain any added protein. The positions of free DNA probe (F) and DNA probe bound in a Rep complex (B) are shown to the right. (B) Enhanced binding of Rep68 to the RRS fragment. Either 1 ng (+) or 2 ng (++) of His-tagged recombinant Rep68 protein (Rep68H) was incubated with a radiolabeled RRS fragment together with the indicated ssDBP. (C) ssDBPs increase binding of a mutant Rep protein that has lost helicase activity. A mutant Rep protein (Y121H/K340H) was incubated in an EMSA with a 32 P-labeled linear DNA fragment containing the RRS, in the presence or absence of the indicated ssDBPs. (D) Complex dissociation was assessed with Rep68H alone or in the presence of Ad-DBP. The Rep68H protein was bound to the RRS fragment and then dissociation was assessed by the addition of increasing amounts of unlabeled competitor DNA to assembled complexes. Binding assays were analyzed by gel electrophoresis and relative binding was quantitated by PhosphorImager analysis of gels.

    Journal: Journal of Virology

    Article Title: The Rep Protein of Adeno-Associated Virus Type 2 Interacts with Single-Stranded DNA-Binding Proteins That Enhance Viral Replication

    doi: 10.1128/JVI.78.1.441-453.2004

    Figure Lengend Snippet: Enhanced binding by Rep proteins is specific and does not require helicase activity. (A) Effect of ssDBPs on binding of MBPRep78 to a linear DNA substrate containing the RRS. The radiolabeled RRS fragment (1,000 cpm) was incubated with MBPRep78 (the numbers at the top indicate the amounts of protein, in micrograms). The ssDBPs were incubated with the probe in the absence (−) or presence (+) of MBPRep78. Binding was enhanced by the HSV-1 ICP8 and RPA proteins but not by the E. coli SSB protein. A constant amount of Rep (indicated by the asterisk below) was assessed for the effect of the ssDBPs. The lane marked “RRS alone” does not contain any added protein. The positions of free DNA probe (F) and DNA probe bound in a Rep complex (B) are shown to the right. (B) Enhanced binding of Rep68 to the RRS fragment. Either 1 ng (+) or 2 ng (++) of His-tagged recombinant Rep68 protein (Rep68H) was incubated with a radiolabeled RRS fragment together with the indicated ssDBP. (C) ssDBPs increase binding of a mutant Rep protein that has lost helicase activity. A mutant Rep protein (Y121H/K340H) was incubated in an EMSA with a 32 P-labeled linear DNA fragment containing the RRS, in the presence or absence of the indicated ssDBPs. (D) Complex dissociation was assessed with Rep68H alone or in the presence of Ad-DBP. The Rep68H protein was bound to the RRS fragment and then dissociation was assessed by the addition of increasing amounts of unlabeled competitor DNA to assembled complexes. Binding assays were analyzed by gel electrophoresis and relative binding was quantitated by PhosphorImager analysis of gels.

    Article Snippet: The E. coli single-stranded DNA-binding protein (SSB) was purchased from Amersham Pharmacia.

    Techniques: Binding Assay, Activity Assay, Incubation, Recombinase Polymerase Amplification, Recombinant, Mutagenesis, Labeling, Nucleic Acid Electrophoresis

    Enhancement of Rep endonucleas e activity by ssDBPs. The trs nicking assay included 32 P-labeled AAV terminal repeat hairpin DNA (1,000 cpm) and either wild-type or mutant purified Rep68H in the presence or absence of the ssDBPs. (A) A representative experiment demonstrating increased endonuclease activity of Rep68H in the presence of ICP8, RPA, and Ad-DBP, but not E. coli SSB. Purified recombinant proteins of wild-type Rep68H (W) or the Y121H/K340H mutant (M) were incubated with the ITR in the hairpin configuration. A titration of wild-type Rep68H showed nicking of the hairpin substrate (S) and release of the cleavage product (P). The mutant failed to nick the double-stranded hairpin ITR. The amount of Rep protein (in nanograms) is indicated above the lanes, and the asterisks below indicate the amounts of wild-type and mutant Rep proteins incubated with the ssDBPs. The amount of each ssDBP included is indicated at the top, in nanograms. Samples of the hairpin (ITR alone) and the ssDBPs in the absence of Rep protein were included as negative controls. (B) Quantitation of endonuclease activities. The amount of nicked product in each reaction was quantitated by PhosphorImager analysis and plotted relative to a constant amount of Rep protein (1 ng). At least three independent reactions were quantitated for each experimental condition.

    Journal: Journal of Virology

    Article Title: The Rep Protein of Adeno-Associated Virus Type 2 Interacts with Single-Stranded DNA-Binding Proteins That Enhance Viral Replication

    doi: 10.1128/JVI.78.1.441-453.2004

    Figure Lengend Snippet: Enhancement of Rep endonucleas e activity by ssDBPs. The trs nicking assay included 32 P-labeled AAV terminal repeat hairpin DNA (1,000 cpm) and either wild-type or mutant purified Rep68H in the presence or absence of the ssDBPs. (A) A representative experiment demonstrating increased endonuclease activity of Rep68H in the presence of ICP8, RPA, and Ad-DBP, but not E. coli SSB. Purified recombinant proteins of wild-type Rep68H (W) or the Y121H/K340H mutant (M) were incubated with the ITR in the hairpin configuration. A titration of wild-type Rep68H showed nicking of the hairpin substrate (S) and release of the cleavage product (P). The mutant failed to nick the double-stranded hairpin ITR. The amount of Rep protein (in nanograms) is indicated above the lanes, and the asterisks below indicate the amounts of wild-type and mutant Rep proteins incubated with the ssDBPs. The amount of each ssDBP included is indicated at the top, in nanograms. Samples of the hairpin (ITR alone) and the ssDBPs in the absence of Rep protein were included as negative controls. (B) Quantitation of endonuclease activities. The amount of nicked product in each reaction was quantitated by PhosphorImager analysis and plotted relative to a constant amount of Rep protein (1 ng). At least three independent reactions were quantitated for each experimental condition.

    Article Snippet: The E. coli single-stranded DNA-binding protein (SSB) was purchased from Amersham Pharmacia.

    Techniques: Activity Assay, Labeling, Mutagenesis, Purification, Recombinase Polymerase Amplification, Recombinant, Incubation, Titration, Quantitation Assay

    YB-1C also forms ssDNA nucleoprotein filaments, unwinds secondary structures and is compatible with mRNA translation. ( A ) AFM images of circular ssDNA (M13) complexed with indicated proteins at saturation. M13 ssDNA, 2 nM; SSB, 1 μM; YB-1, 0.5 μM; aa 1–219, 0.7 μM; aa 1–180 (YB-1C), 2 μM. Lower right panel: contour length of ssDNA or mRNA nucleoprotein filaments. N = 10. Blue bar: mean. ( B ) A DNA construct consisting of two circular ssDNA (1500 and 5000 nt) separated by a dsDNA stalk (300 bp) complexed with either YB-1 or YB-1C. ssDNA, 0.5 nM; YB-1, 0.5 μM; aa 1–180 (YB-1C), 2 μM. ( C ) Left panel: electrophoretic mobility of mRNA or ssDNA in the presence of different proteins. The presence of YB-1C at saturation decreases EtBr fluorescence of the mRNA band, as observed for ssDNA nucleoprotein filament with Escherichia coli SSB. Right panel: quantification of ethidium fluorescence of three replicates under the same condition. ( D ) In vitro translation assay in RRL. mRNA was pre-incubated with proteins for 10 min before addition in RRL for 10 min. Note the inhibition of mRNA translation by YB-1 but not by YB-1C. mRNA: 4 nM. Anti-YB-1 and anti-luciferase primary antibodies. Both endogenous and overexpressed YB-1 constructs are recognized by the anti-YB-1 antibody (see also Supplementary Figure S1 ).

    Journal: Nucleic Acids Research

    Article Title: YB-1, an abundant core mRNA-binding protein, has the capacity to form an RNA nucleoprotein filament: a structural analysis

    doi: 10.1093/nar/gky1303

    Figure Lengend Snippet: YB-1C also forms ssDNA nucleoprotein filaments, unwinds secondary structures and is compatible with mRNA translation. ( A ) AFM images of circular ssDNA (M13) complexed with indicated proteins at saturation. M13 ssDNA, 2 nM; SSB, 1 μM; YB-1, 0.5 μM; aa 1–219, 0.7 μM; aa 1–180 (YB-1C), 2 μM. Lower right panel: contour length of ssDNA or mRNA nucleoprotein filaments. N = 10. Blue bar: mean. ( B ) A DNA construct consisting of two circular ssDNA (1500 and 5000 nt) separated by a dsDNA stalk (300 bp) complexed with either YB-1 or YB-1C. ssDNA, 0.5 nM; YB-1, 0.5 μM; aa 1–180 (YB-1C), 2 μM. ( C ) Left panel: electrophoretic mobility of mRNA or ssDNA in the presence of different proteins. The presence of YB-1C at saturation decreases EtBr fluorescence of the mRNA band, as observed for ssDNA nucleoprotein filament with Escherichia coli SSB. Right panel: quantification of ethidium fluorescence of three replicates under the same condition. ( D ) In vitro translation assay in RRL. mRNA was pre-incubated with proteins for 10 min before addition in RRL for 10 min. Note the inhibition of mRNA translation by YB-1 but not by YB-1C. mRNA: 4 nM. Anti-YB-1 and anti-luciferase primary antibodies. Both endogenous and overexpressed YB-1 constructs are recognized by the anti-YB-1 antibody (see also Supplementary Figure S1 ).

    Article Snippet: Recombinant E. coli single stranded DNA binding (SSB) protein was purchased from Abcam (ab123224).

    Techniques: Construct, Fluorescence, In Vitro, Incubation, Inhibition, Luciferase

    Ideication of the UV cross-linked amino acid residue in SSB by nanoelectrospray tandem mass spectrometry of the peptide–DNA heteroconjugate. ( A ) Nanoelectrospray mass spectrum obtained from the Oligo R3 eluate after tryptic digest, IMAC purification, and phosphodiesterase I digest. The major ion signal is attributable to a triply charged species at m/z 779.62. ( B ) First MS/MS fragmentation regime: product ion spectrum of the triply charged species at m/z 779.62 at a Q 0 setting of 40 V (about 60 eV collision energy in LRF). The major fragments corresponding to the fragmentation of a trinucleotide with the sequence TpGpX are labeled. The fragments corresponding to the loss of the former 5-iodouracil are marked with triangles. No peptide backbone cleavage products are observable. ( C ) Second MS/MS fragmentation regime: product ion spectrum of the same species at a Q 0 setting of 60 V (about 120 eV collision energy in LRF). The region above m/z 300 is enlarged 46-fold showing fragments deriving from the cleavages of the amide bond. The fragment ions y 8 and y 9 unambiguously demonstrate that W88 was cross-linked to the DNA. Sequence specific fragment ions are labeled. ( D ) in bold face; amino acid one-letter code underlined in bold face. The lower part shows the fragmentation of the peptide at a collision energy of about 120 eV. The peptide fragment ions are labeled according to Biemann (1988). ( E ) Product ion spectrum of the triply charged peptide–trinucleotide heteroconjugate at m/z 615.5, revealing Trp-54 to be the second UV-cross-linked amino acid residue.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: Tryptophans 54 and 88 of E. coli SSB cross-link to DNA

    doi:

    Figure Lengend Snippet: Ideication of the UV cross-linked amino acid residue in SSB by nanoelectrospray tandem mass spectrometry of the peptide–DNA heteroconjugate. ( A ) Nanoelectrospray mass spectrum obtained from the Oligo R3 eluate after tryptic digest, IMAC purification, and phosphodiesterase I digest. The major ion signal is attributable to a triply charged species at m/z 779.62. ( B ) First MS/MS fragmentation regime: product ion spectrum of the triply charged species at m/z 779.62 at a Q 0 setting of 40 V (about 60 eV collision energy in LRF). The major fragments corresponding to the fragmentation of a trinucleotide with the sequence TpGpX are labeled. The fragments corresponding to the loss of the former 5-iodouracil are marked with triangles. No peptide backbone cleavage products are observable. ( C ) Second MS/MS fragmentation regime: product ion spectrum of the same species at a Q 0 setting of 60 V (about 120 eV collision energy in LRF). The region above m/z 300 is enlarged 46-fold showing fragments deriving from the cleavages of the amide bond. The fragment ions y 8 and y 9 unambiguously demonstrate that W88 was cross-linked to the DNA. Sequence specific fragment ions are labeled. ( D ) in bold face; amino acid one-letter code underlined in bold face. The lower part shows the fragmentation of the peptide at a collision energy of about 120 eV. The peptide fragment ions are labeled according to Biemann (1988). ( E ) Product ion spectrum of the triply charged peptide–trinucleotide heteroconjugate at m/z 615.5, revealing Trp-54 to be the second UV-cross-linked amino acid residue.

    Article Snippet: Recombinant E. coli single-strand DNA-binding protein (SSB) was purchased from Stratagene.

    Techniques: Mass Spectrometry, Purification, Sequencing, Labeling

    Isolation of the DNA–peptide conjugate by urea PAGE and analysis by MALDI-MS. The reaction was spiked with My9IU–DNA labeled at its 5`-terminus with [ 32 P]-phosphate. ( A ) Autoradiography of an urea PAGE after trypsinolysis of the SSB–DNA cross-link reaction. Bands attributable to unreacted DNA, peptide–DNA conjugate, and undigested SSB–DNA are indicated. ( B ) MALDI mass spectrum of the peptide–DNA heteroconjugate after elution from the gel band (indicated in [ A ]) showing ion signals attributable to DNA–T 85–96 (TRKWTDQSGQDR) at m/z 7308.1 and to DNA –T 50–56 (EQTEWHR) at m/z 6816.0. ( C ) Control: MALDI mass spectrum of unreacted My9IU, showing singly, doubly, and triply charged ion species. The ion signals marked with asterisks in ( B ) and ( C ) correspond to a DNA synthesis aact.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: Tryptophans 54 and 88 of E. coli SSB cross-link to DNA

    doi:

    Figure Lengend Snippet: Isolation of the DNA–peptide conjugate by urea PAGE and analysis by MALDI-MS. The reaction was spiked with My9IU–DNA labeled at its 5`-terminus with [ 32 P]-phosphate. ( A ) Autoradiography of an urea PAGE after trypsinolysis of the SSB–DNA cross-link reaction. Bands attributable to unreacted DNA, peptide–DNA conjugate, and undigested SSB–DNA are indicated. ( B ) MALDI mass spectrum of the peptide–DNA heteroconjugate after elution from the gel band (indicated in [ A ]) showing ion signals attributable to DNA–T 85–96 (TRKWTDQSGQDR) at m/z 7308.1 and to DNA –T 50–56 (EQTEWHR) at m/z 6816.0. ( C ) Control: MALDI mass spectrum of unreacted My9IU, showing singly, doubly, and triply charged ion species. The ion signals marked with asterisks in ( B ) and ( C ) correspond to a DNA synthesis aact.

    Article Snippet: Recombinant E. coli single-strand DNA-binding protein (SSB) was purchased from Stratagene.

    Techniques: Isolation, Polyacrylamide Gel Electrophoresis, Mass Spectrometry, Labeling, Autoradiography, DNA Synthesis

    SDS-PAGE and differential peptide mass mapping of SSB–DNA heteroconjugate. ( A ) SDS-PAGE separation of protein–DNA conjugate from unreacted protein (visualized by colloidal Coomassie staining). ( B ) Peptide mass map obtained after in-gel trypsinolysis of SSB–DNA cross-link. ( C ) Control: peptide mass map of unreacted SSB, in-gel digested with trypsin. ( D ) Peptide mass map obtained after in-gel digestion of SSB–DNA cross-link with endoproteinase GluC. ( E ) Control: peptide mass map of unreacted SSB, in-gel digested with endoproteinase GluC. Peptides, which are present in the control but not in the digest of the heteroconjugate, are marked with arrows.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: Tryptophans 54 and 88 of E. coli SSB cross-link to DNA

    doi:

    Figure Lengend Snippet: SDS-PAGE and differential peptide mass mapping of SSB–DNA heteroconjugate. ( A ) SDS-PAGE separation of protein–DNA conjugate from unreacted protein (visualized by colloidal Coomassie staining). ( B ) Peptide mass map obtained after in-gel trypsinolysis of SSB–DNA cross-link. ( C ) Control: peptide mass map of unreacted SSB, in-gel digested with trypsin. ( D ) Peptide mass map obtained after in-gel digestion of SSB–DNA cross-link with endoproteinase GluC. ( E ) Control: peptide mass map of unreacted SSB, in-gel digested with endoproteinase GluC. Peptides, which are present in the control but not in the digest of the heteroconjugate, are marked with arrows.

    Article Snippet: Recombinant E. coli single-strand DNA-binding protein (SSB) was purchased from Stratagene.

    Techniques: SDS Page, Staining

    DdrC stimulates DNA annealing. Kinetics of two complementary 67-mer oligonucleotides annealing in the absence (w/o protein) or the presence of DdrC, T4 gp32 or SSB using a DAPI fluorescence-based method. The 67-mer oligonucleotide (200 nM) was mixed in 1 ml of reaction buffer with 0.2 μM DdrC protein, or 0.1 μM T4 gp32, or 0.1 μM SSB from E . coli prior to addition of the reverse oligonucleotide. The extent of DNA annealing is defined as follows: (observed fluorescence—67-mer ssDNA fluorescence) x 100 / 67-mer ds DNA fluorescence.

    Journal: PLoS ONE

    Article Title: In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

    doi: 10.1371/journal.pone.0177751

    Figure Lengend Snippet: DdrC stimulates DNA annealing. Kinetics of two complementary 67-mer oligonucleotides annealing in the absence (w/o protein) or the presence of DdrC, T4 gp32 or SSB using a DAPI fluorescence-based method. The 67-mer oligonucleotide (200 nM) was mixed in 1 ml of reaction buffer with 0.2 μM DdrC protein, or 0.1 μM T4 gp32, or 0.1 μM SSB from E . coli prior to addition of the reverse oligonucleotide. The extent of DNA annealing is defined as follows: (observed fluorescence—67-mer ssDNA fluorescence) x 100 / 67-mer ds DNA fluorescence.

    Article Snippet: PhiX174 ssDNA covered with E . coli Single Strand DNA Binding Protein (SSB, 1 μM) (Sigma) was used as control.

    Techniques: Fluorescence

    DdrC binds to ssDNA and dsDNA with a preference for ssDNA. A Binding of recombinant DdrC to plasmid or viral DNA analyzed by EMSA. 200 ng of supercoiled or linear pBR322 DNA as well as 200 ng of RFI or single-stranded DNA of phiX174 virion (31 μM nucleotides of each DNA) were incubated with increasing concentrations of DdrC as indicated in the figure. DNA-protein complexes were separated in 1.2% agarose gels. Products loaded in the right lane of the left panel were treated with SDS and proteinase K. sc: supercoiled dsDNA, oc: open circle dsDNA, Li: linear dsDNA. B Binding of DdrC to oligonucleotides. Increasing concentrations of DdrC were incubated with 3.3 nM of a single-stranded (ss) 67-mer fluorescent oligonucleotide (left panel) or 3.3 nM of the corresponding ds oligonucleotide (right panel). The products of the reactions were separated in 6% native polyacrylamide gels. Lanes C: DNA control without DdrC.

    Journal: PLoS ONE

    Article Title: In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

    doi: 10.1371/journal.pone.0177751

    Figure Lengend Snippet: DdrC binds to ssDNA and dsDNA with a preference for ssDNA. A Binding of recombinant DdrC to plasmid or viral DNA analyzed by EMSA. 200 ng of supercoiled or linear pBR322 DNA as well as 200 ng of RFI or single-stranded DNA of phiX174 virion (31 μM nucleotides of each DNA) were incubated with increasing concentrations of DdrC as indicated in the figure. DNA-protein complexes were separated in 1.2% agarose gels. Products loaded in the right lane of the left panel were treated with SDS and proteinase K. sc: supercoiled dsDNA, oc: open circle dsDNA, Li: linear dsDNA. B Binding of DdrC to oligonucleotides. Increasing concentrations of DdrC were incubated with 3.3 nM of a single-stranded (ss) 67-mer fluorescent oligonucleotide (left panel) or 3.3 nM of the corresponding ds oligonucleotide (right panel). The products of the reactions were separated in 6% native polyacrylamide gels. Lanes C: DNA control without DdrC.

    Article Snippet: PhiX174 ssDNA covered with E . coli Single Strand DNA Binding Protein (SSB, 1 μM) (Sigma) was used as control.

    Techniques: Binding Assay, Recombinant, Plasmid Preparation, Incubation

    Visualization of DdrC-DNA complexes by transmission electron microscopy. A PhiX174 ssDNA (1.4 nM, 7.5 μM nucleotides) was incubated with 1 μM (panels b-d) or 2 μM (panels f-h) of DdrC. Panel a: phiX174 ssDNA control without DdrC. Panel e: Interaction of E . coli SSB protein (1 μM) with ssDNA. Magnification = 85,000. B Supercoiled pBR322 DNA (1.7 nM, 7.5 μM base pairs) incubated with 1 μM (panel b and c) or 2 μM (panel d) of DdrC. Panel a: pBR322 DNA control without protein. Magnification = 85,000. Some“bridge” structures, forming loops or kinks, are indicated by arrows.

    Journal: PLoS ONE

    Article Title: In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

    doi: 10.1371/journal.pone.0177751

    Figure Lengend Snippet: Visualization of DdrC-DNA complexes by transmission electron microscopy. A PhiX174 ssDNA (1.4 nM, 7.5 μM nucleotides) was incubated with 1 μM (panels b-d) or 2 μM (panels f-h) of DdrC. Panel a: phiX174 ssDNA control without DdrC. Panel e: Interaction of E . coli SSB protein (1 μM) with ssDNA. Magnification = 85,000. B Supercoiled pBR322 DNA (1.7 nM, 7.5 μM base pairs) incubated with 1 μM (panel b and c) or 2 μM (panel d) of DdrC. Panel a: pBR322 DNA control without protein. Magnification = 85,000. Some“bridge” structures, forming loops or kinks, are indicated by arrows.

    Article Snippet: PhiX174 ssDNA covered with E . coli Single Strand DNA Binding Protein (SSB, 1 μM) (Sigma) was used as control.

    Techniques: Transmission Assay, Electron Microscopy, Incubation

    DdrC protects DNA against degradation by nucleases. Protection of supercoiled pBR322 plasmid (3.5 nM) from DNase I activity (0.1 U) (panel a), linear pBR322 (3.5 nM) from Exonuclease III activity (200 U) (panel b) and phiX174 ssDNA (5.9 nM) from Mung Bean Nuclease activity (1 U) (panel c) by 7 μM, 7 μM, and 2 μM DdrC, respectively. Lanes C: DNA controls without protein. Lanes 1: DNA incubation with nuclease alone. Lanes 2: DNA incubation with DdrC alone. Lanes 3: DNA pre-incubated with DdrC 15 min at 4°C before addition of nuclease. Lanes 4: Reaction products corresponding to lane 3 were further treated with Proteinase K/SDS. Panel a, lane 5: DdrC and DNase I were simultaneously incubated with supercoiled DNA before treatment with Proteinase K/SDS.

    Journal: PLoS ONE

    Article Title: In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

    doi: 10.1371/journal.pone.0177751

    Figure Lengend Snippet: DdrC protects DNA against degradation by nucleases. Protection of supercoiled pBR322 plasmid (3.5 nM) from DNase I activity (0.1 U) (panel a), linear pBR322 (3.5 nM) from Exonuclease III activity (200 U) (panel b) and phiX174 ssDNA (5.9 nM) from Mung Bean Nuclease activity (1 U) (panel c) by 7 μM, 7 μM, and 2 μM DdrC, respectively. Lanes C: DNA controls without protein. Lanes 1: DNA incubation with nuclease alone. Lanes 2: DNA incubation with DdrC alone. Lanes 3: DNA pre-incubated with DdrC 15 min at 4°C before addition of nuclease. Lanes 4: Reaction products corresponding to lane 3 were further treated with Proteinase K/SDS. Panel a, lane 5: DdrC and DNase I were simultaneously incubated with supercoiled DNA before treatment with Proteinase K/SDS.

    Article Snippet: PhiX174 ssDNA covered with E . coli Single Strand DNA Binding Protein (SSB, 1 μM) (Sigma) was used as control.

    Techniques: Plasmid Preparation, Activity Assay, Incubation