Structured Review

Double Helix dna double helix
( a ) Selected contact mode <t>HS-AFM</t> frame of disrupted Emiliania huxleyi Virus 86 spilling its genomic content captured at 0.5 s per frame by the authors (max height 37.1 nm); ( b ) Selected AFM image of PBCV-1 <t>DNA</t> pre proteinase K treatment, used with permission and adapted from Wulfmeyer et al. [ 106 ] (scale bar 100 nm, max height 2.4 nm). Arrows show ([a]-putative) DNA associated proteins.
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Images

1) Product Images from "Algal Viruses: The (Atomic) Shape of Things to Come"

Article Title: Algal Viruses: The (Atomic) Shape of Things to Come

Journal: Viruses

doi: 10.3390/v10090490

( a ) Selected contact mode HS-AFM frame of disrupted Emiliania huxleyi Virus 86 spilling its genomic content captured at 0.5 s per frame by the authors (max height 37.1 nm); ( b ) Selected AFM image of PBCV-1 DNA pre proteinase K treatment, used with permission and adapted from Wulfmeyer et al. [ 106 ] (scale bar 100 nm, max height 2.4 nm). Arrows show ([a]-putative) DNA associated proteins.
Figure Legend Snippet: ( a ) Selected contact mode HS-AFM frame of disrupted Emiliania huxleyi Virus 86 spilling its genomic content captured at 0.5 s per frame by the authors (max height 37.1 nm); ( b ) Selected AFM image of PBCV-1 DNA pre proteinase K treatment, used with permission and adapted from Wulfmeyer et al. [ 106 ] (scale bar 100 nm, max height 2.4 nm). Arrows show ([a]-putative) DNA associated proteins.

Techniques Used:

2) Product Images from "Structural and Functional Interactions of Transcription Factor (TF) IIA with TFIIE and TFIIF in Transcription Initiation by RNA Polymerase II"

Article Title: Structural and Functional Interactions of Transcription Factor (TF) IIA with TFIIE and TFIIF in Transcription Initiation by RNA Polymerase II

Journal: The Journal of biological chemistry

doi: 10.1074/jbc.M106422200

Photo-cross-linking of rTFIIA on the AdMLP A, photo-cross-linking experiments with photoprobes −39/−40 and +26 were performed in the presence of rTFIIA ( α/β and γ ). The position of TFIIA α/β and TFIIA γ are indicated. B, schematic representation of promoter contacts by TFIIA in the context of the TBP-TFIIA-promoter complex and the TBP-TFIIA-TFIIB-TFIIF-RNAPII-TFIIE-promoter complex in which the DNA adopts a wrapped structure. Positions −39/−40, +26 and +1 are indicated. Only TBP, TFIIA, and RNAPII are represented to simplify the diagram.
Figure Legend Snippet: Photo-cross-linking of rTFIIA on the AdMLP A, photo-cross-linking experiments with photoprobes −39/−40 and +26 were performed in the presence of rTFIIA ( α/β and γ ). The position of TFIIA α/β and TFIIA γ are indicated. B, schematic representation of promoter contacts by TFIIA in the context of the TBP-TFIIA-promoter complex and the TBP-TFIIA-TFIIB-TFIIF-RNAPII-TFIIE-promoter complex in which the DNA adopts a wrapped structure. Positions −39/−40, +26 and +1 are indicated. Only TBP, TFIIA, and RNAPII are represented to simplify the diagram.

Techniques Used:

Association of RAP74 with a TBP-TFIIA-promoter complex Gel mobility shift assays were performed using a radiolabeled DNA fragment comprising the AdMLP in the presence of TBP alone, TBP, and TFIIA and TBP, TFIIA and various fragments of RAP74 ( RAP74-(1–517;wt ), RAP74-(1–75), RAP74-(1–136), RAP74-(363–409), and RAP74-(363–444)). The positions of the TBP ( T ), TBP-TFIIA ( T-A ), and TBP-TFIIA-RAP74 ( T-A-RAP74 ) complexes and that of the free probe are indicated.
Figure Legend Snippet: Association of RAP74 with a TBP-TFIIA-promoter complex Gel mobility shift assays were performed using a radiolabeled DNA fragment comprising the AdMLP in the presence of TBP alone, TBP, and TFIIA and TBP, TFIIA and various fragments of RAP74 ( RAP74-(1–517;wt ), RAP74-(1–75), RAP74-(1–136), RAP74-(363–409), and RAP74-(363–444)). The positions of the TBP ( T ), TBP-TFIIA ( T-A ), and TBP-TFIIA-RAP74 ( T-A-RAP74 ) complexes and that of the free probe are indicated.

Techniques Used: Mobility Shift

3) Product Images from "Werner syndrome as a hereditary risk factor for exocrine pancreatic cancer"

Article Title: Werner syndrome as a hereditary risk factor for exocrine pancreatic cancer

Journal: Cancer Biology & Therapy

doi: 10.4161/cbt.10.5.12763

A schematic diagram of WRN with the loss-of-function mutations identified in the 43-year-old patient who developed pancreatic adenocarcinoma. The WRN gene encodes a helicase of the RecQ family containing an exonuclease domain, and also possesses a transactivation domain (TAD), RecQ conserved domain (RQC), helicase and RNase D C-terminus domain (HRDC) and nuclear localization sequence (NLS). This patient possessed mutations 009 and 039 as defined by the Werner International Registry. Mutation 009 caused a stop codon at exon 9 through a cytosine-to-thymine transition (C1105T), leading to the substitution of arginine by a stop codon (R369stop), and resulting in the truncation of the helicase and exonuclease domains. Mutation 039 caused a guanine-to-cytosine transversion (G3139-1C) at the junction of intron 25 and exon 26, leading to the substitution of phenylalanine for glycine (G1047F), and resulting in a frameshift and insertion of a stop codon 14 amino acids distal to the nucleotide change that abolished the NLS domain. AA, amino acid; BP, DNA base pair.
Figure Legend Snippet: A schematic diagram of WRN with the loss-of-function mutations identified in the 43-year-old patient who developed pancreatic adenocarcinoma. The WRN gene encodes a helicase of the RecQ family containing an exonuclease domain, and also possesses a transactivation domain (TAD), RecQ conserved domain (RQC), helicase and RNase D C-terminus domain (HRDC) and nuclear localization sequence (NLS). This patient possessed mutations 009 and 039 as defined by the Werner International Registry. Mutation 009 caused a stop codon at exon 9 through a cytosine-to-thymine transition (C1105T), leading to the substitution of arginine by a stop codon (R369stop), and resulting in the truncation of the helicase and exonuclease domains. Mutation 039 caused a guanine-to-cytosine transversion (G3139-1C) at the junction of intron 25 and exon 26, leading to the substitution of phenylalanine for glycine (G1047F), and resulting in a frameshift and insertion of a stop codon 14 amino acids distal to the nucleotide change that abolished the NLS domain. AA, amino acid; BP, DNA base pair.

Techniques Used: Sequencing, Mutagenesis

4) Product Images from "Interaction of Proliferation Cell Nuclear Antigen (PCNA) with c-Abl in Cell Proliferation and Response to DNA Damages in Breast Cancer"

Article Title: Interaction of Proliferation Cell Nuclear Antigen (PCNA) with c-Abl in Cell Proliferation and Response to DNA Damages in Breast Cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0029416

c-Abl enhances chromatin association and PCNA foci formation in response to DNA damage. A. MDA-MB-231/shAbl and MDA-MB-231/shCtrl cells were exposed to 10 Gy of IR. Following one hour of incubation, the cells were extracted with 0.5% of Triton X-100. Levels of PCNA in the soluble and insoluble fractions were examined by western analysis. Expression of α-tubulin and histone H3 was assessed as markers of the soluble and insoluble fractions, respectively. B. c-Abl is important in the formation of PCNA nuclear foci. MDA-MB-231/shAbl and MDA-MB-231/shCtrl cells were mock-treated or irradiated with 10 Gy of IR followed by incubation for one hour. Cells were then fixed with methanol and stained with an anti-PCNA antibody. C. To evaluate the number of foci-positive cells, five independent fields representing each treatment were counted. The experiment was repeated two times, and the results of the two trials were consistent.
Figure Legend Snippet: c-Abl enhances chromatin association and PCNA foci formation in response to DNA damage. A. MDA-MB-231/shAbl and MDA-MB-231/shCtrl cells were exposed to 10 Gy of IR. Following one hour of incubation, the cells were extracted with 0.5% of Triton X-100. Levels of PCNA in the soluble and insoluble fractions were examined by western analysis. Expression of α-tubulin and histone H3 was assessed as markers of the soluble and insoluble fractions, respectively. B. c-Abl is important in the formation of PCNA nuclear foci. MDA-MB-231/shAbl and MDA-MB-231/shCtrl cells were mock-treated or irradiated with 10 Gy of IR followed by incubation for one hour. Cells were then fixed with methanol and stained with an anti-PCNA antibody. C. To evaluate the number of foci-positive cells, five independent fields representing each treatment were counted. The experiment was repeated two times, and the results of the two trials were consistent.

Techniques Used: Multiple Displacement Amplification, Incubation, Western Blot, Expressing, Irradiation, Staining

Growth inhibition of BT474 cells by targeting Y211 phosphorylation of PCNA. A. Cells mock-treated with vehicle alone or treated with the scramble or Y211F peptides (15 µM) were subjected to flow cytometry analysis. The percentages of cells in the G1, S, and G2/M phases were plotted. B. DNA synthesis activity in the treated cells was determined by a colorimetric BrdU-incorporation analysis. For each data point, the amount of incorporated BrdU was normalized to the percentage of viable cells, as determined by a side-by-side MTT assay. C. Depletion of c-Abl decreases sensitivity to Y211F peptide-mediated growth inhibition. A derivative of BT474 cells harboring an shRNA against c-Abl (BT474/shAbl ) or luciferase (BT474/shLuc) were treated with 10 µM Y211F peptide or the scramble peptide for 48 h. Surviving cells were then assessed by MTT assay and the results were plotted. *, P
Figure Legend Snippet: Growth inhibition of BT474 cells by targeting Y211 phosphorylation of PCNA. A. Cells mock-treated with vehicle alone or treated with the scramble or Y211F peptides (15 µM) were subjected to flow cytometry analysis. The percentages of cells in the G1, S, and G2/M phases were plotted. B. DNA synthesis activity in the treated cells was determined by a colorimetric BrdU-incorporation analysis. For each data point, the amount of incorporated BrdU was normalized to the percentage of viable cells, as determined by a side-by-side MTT assay. C. Depletion of c-Abl decreases sensitivity to Y211F peptide-mediated growth inhibition. A derivative of BT474 cells harboring an shRNA against c-Abl (BT474/shAbl ) or luciferase (BT474/shLuc) were treated with 10 µM Y211F peptide or the scramble peptide for 48 h. Surviving cells were then assessed by MTT assay and the results were plotted. *, P

Techniques Used: Inhibition, Flow Cytometry, Cytometry, DNA Synthesis, Activity Assay, BrdU Incorporation Assay, MTT Assay, shRNA, Luciferase

5) Product Images from "The Deletion of rnhB in Mycobacterium smegmatis Does Not Affect the Level of RNase HII Substrates or Influence Genome Stability"

Article Title: The Deletion of rnhB in Mycobacterium smegmatis Does Not Affect the Level of RNase HII Substrates or Influence Genome Stability

Journal: PLoS ONE

doi: 10.1371/journal.pone.0115521

Alkaline hydrolysis of the genomic DNA. DNA was isolated from the ∆ rnhB mutants and M. smegmatis mc 2 155. The strains were grown in 7H9 medium supplemented with OADC. The DNA samples were treated with either NaOH or NaCl as a control. The fragmentation of the samples was visualized on alkaline agarose gels. Lanes 1a) GeneRuler 1-kb DNA Ladder, 2a) M. smegmatis mc 2 155 control DNA, 3a) ∆ rnhB mutant control DNA, 1b) GeneRuler 1-kb DNA Ladder, 2b) M. smegmatis mc 2 155 DNA hydrolyzed with NaOH, and 3b) ∆ rnhB mutant DNA hydrolyzed with NaOH. The level of ribonucleotide incorporated in the DNA of both strains was similar, as we did not observe differences in fragmentation of genomic DNA.
Figure Legend Snippet: Alkaline hydrolysis of the genomic DNA. DNA was isolated from the ∆ rnhB mutants and M. smegmatis mc 2 155. The strains were grown in 7H9 medium supplemented with OADC. The DNA samples were treated with either NaOH or NaCl as a control. The fragmentation of the samples was visualized on alkaline agarose gels. Lanes 1a) GeneRuler 1-kb DNA Ladder, 2a) M. smegmatis mc 2 155 control DNA, 3a) ∆ rnhB mutant control DNA, 1b) GeneRuler 1-kb DNA Ladder, 2b) M. smegmatis mc 2 155 DNA hydrolyzed with NaOH, and 3b) ∆ rnhB mutant DNA hydrolyzed with NaOH. The level of ribonucleotide incorporated in the DNA of both strains was similar, as we did not observe differences in fragmentation of genomic DNA.

Techniques Used: Isolation, Mutagenesis

Related Articles

Binding Assay:

Article Title: Identification and molecular characterization of an Alba-family protein from human malaria parasite Plasmodium falciparum
Article Snippet: .. Loops L1, L3 and L5 play an important role in DNA binding, whereas surface exposed lysines present in KKP motif in the loop L1 (K22 and K23) are found in very close proximity with the DNA double helix ( D). .. Lysine (K) 56 and lysine (K) 67 of helix α2 are also located beneath the docked DNA, hence may be involved in DNA binding.

Article Title: Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands
Article Snippet: Even though this mutated protein does not contain any ligand in its binding pocket, X-ray data revealed that the distance separating the P59 (Cα ) of each HTH motifs is 42.3 Å ( d). .. Binding of EthRG106W to DNA With its DNA recognition helices α3 and α3′ separated by 41.5 Å, the G106W variant of EthR was predicted to be unable to bind its DNA operator, as this distance amply exceeds the helical periodicity of the B-DNA double helix (34 Å per turn of the helix). ..

Article Title: Streptococcus pyogenes pSM19035 requires dynamic assembly of ATP-bound ParA and ParB on parS DNA during plasmid segregation
Article Snippet: In presence of ATP•Mg+2 , δ2 (100 nM) assembled to form discrete clusters on ∼85% of the DNA molecules (n = 200) at random locations ( B), whereas ∼40% of the DNA molecules (n = 250) that were incubated only with ω2 showed clusters of ω2 bound to parS on the plasmid ( C). .. The parS DNA region on linear DNA was not significantly distorted by ω2 binding, consistent with the prediction based on crystal structures that protein ω2 would wrap around parS sites without significantly bending the DNA double helix [ , C]. ..

Variant Assay:

Article Title: Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands
Article Snippet: Even though this mutated protein does not contain any ligand in its binding pocket, X-ray data revealed that the distance separating the P59 (Cα ) of each HTH motifs is 42.3 Å ( d). .. Binding of EthRG106W to DNA With its DNA recognition helices α3 and α3′ separated by 41.5 Å, the G106W variant of EthR was predicted to be unable to bind its DNA operator, as this distance amply exceeds the helical periodicity of the B-DNA double helix (34 Å per turn of the helix). ..

Fluorescence:

Article Title: Sum rules and determination of exciton coupling using absorption and circular dichroism spectra of biological polymers
Article Snippet: .. Onidas D, Gustavsson T, Lazzarotto E, Markovitsi D. Fluorescence of the DNA double helix (dA)20 (dT)20 studied by femtosecond spectroscopy—Effect of the duplex size on the properties of the excited states. ..

Spectroscopy:

Article Title: Sum rules and determination of exciton coupling using absorption and circular dichroism spectra of biological polymers
Article Snippet: .. Onidas D, Gustavsson T, Lazzarotto E, Markovitsi D. Fluorescence of the DNA double helix (dA)20 (dT)20 studied by femtosecond spectroscopy—Effect of the duplex size on the properties of the excited states. ..

other:

Article Title: Characterizing micro-to-millisecond chemical exchange in nucleic acids using offresonance R1ρ relaxation dispersion
Article Snippet: Watson-Crick to Hoogsteen exchange in DNA and RNA duplexes As stated throughout this review, application of off-resonance R 1ρ RD methodology resulted in the discovery that in the DNA double-helix, A-T and G-C Watson-Crick BPs continually undergo exchange with their Hoogsteen counterparts.

Activation Assay:

Article Title: Effects of Replication and Transcription on DNA Structure-Related Genetic Instability
Article Snippet: .. Many repetitive sequences in the human genome can adopt conformations that differ from the canonical B-DNA double helix (i.e., non-B DNA), and can impact important biological processes such as DNA replication, transcription, recombination, telomere maintenance, viral integration, transposome activation, DNA damage and repair. ..

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    Double Helix dna double helix conformation
    Interaction of BCDX2 complex with single-stranded regions in duplex <t>DNA.</t> ( A – C ) Electron microscopic visualization of complexes formed between BCDX2 and tailed duplex DNA ( A ) or gapped circular duplex DNA ( B ). The bound single-stranded regions are indicated by white arrows. ( C ) Control indicating the failure of BCDX2 to bind linear duplex DNA. ( D ) Juxtaposition of BCDX2 complex and <t>RAD51</t> on gapped circular DNA. The DNA was preincubated with BCDX2 complex (90 nM) for 5 min and then supplemented with RAD51 (0.3 μM). After 5 min at 37°C, the products were visualized by electron microscopy. The white arrow indicates a RAD51 filament, and the black arrow indicates the BCDX2–DNA complexes.
    Dna Double Helix Conformation, supplied by Double Helix, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Double Helix dna double helix
    A. Schematic diagram showing DnaI-mediated loading of the G. kaustophilus ring helicase, DnaC. Monomers of DnaI (green) upon binding to <t>ATP</t> assemble into a hexameric ring around single-stranded <t>DNA.</t> The N-terminal tier of the DnaI ring interacts with the C-terminal tier of the helicase (brown) recruiting and opening up the helicase ring. Single-stranded DNA passes into the central channel of the ring. Binding of the DNA to the internal surface of the central channel stimulates the helicase activity and induces dissociation of DnaI ( Tsai et al ., 2009 ). B. Loading of helicase-primase bifunctional proteins. A helicase-primase protein initially interacts with the single-stranded DNA via its primase domains inducing an opening of the helicase ring with the DNA passing through the gap into the central channel. Binding of the DNA in the central cavity induces ring closure and activates the helicase-primase protein. The T7 gp4 primase-helicase crystallized as a heptamer (the seven subunits are coloured differently in the side view of the structure shown) but the translocating species along single-stranded DNA is believed to be the hexamer ( Toth et al ., 2003 ).
    Dna Double Helix, supplied by Double Helix, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Double Helix helicase
    Archaeal/eukaryotic <t>helicase</t> interacting with DNA. ( A ) DNA opening and unwinding by the Mcm2-7 helicase: the two motor domains that move along the DNA are at opposite ends of a DNA-loaded helicase double ring, with the N-terminal DNA-binding domains (NTD) in the middle. Froelich, Kang et al. propose that the motor domains push the duplex DNA towards the middle of the helicase, hence promoting melting of the DNA at the origin of replication and trapping of the leading strand template by the NTD. The rings then separate and travel in opposite directions, with one ring sliding along each of the leading strand templates from the two replication forks. ( B ) Crystal structure of the MCM helicase (orange) bound to single-stranded DNA (light blue). The DNA circles around the MCM central channel in a clockwise direction (when travelling from the 5′-end to 3′-end and viewing the ring from its C-terminal face).
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    Image Search Results


    Interaction of BCDX2 complex with single-stranded regions in duplex DNA. ( A – C ) Electron microscopic visualization of complexes formed between BCDX2 and tailed duplex DNA ( A ) or gapped circular duplex DNA ( B ). The bound single-stranded regions are indicated by white arrows. ( C ) Control indicating the failure of BCDX2 to bind linear duplex DNA. ( D ) Juxtaposition of BCDX2 complex and RAD51 on gapped circular DNA. The DNA was preincubated with BCDX2 complex (90 nM) for 5 min and then supplemented with RAD51 (0.3 μM). After 5 min at 37°C, the products were visualized by electron microscopy. The white arrow indicates a RAD51 filament, and the black arrow indicates the BCDX2–DNA complexes.

    Journal: Genes & Development

    Article Title: Identification and purification of two distinct complexes containing the five RAD51 paralogs

    doi: 10.1101/gad.947001

    Figure Lengend Snippet: Interaction of BCDX2 complex with single-stranded regions in duplex DNA. ( A – C ) Electron microscopic visualization of complexes formed between BCDX2 and tailed duplex DNA ( A ) or gapped circular duplex DNA ( B ). The bound single-stranded regions are indicated by white arrows. ( C ) Control indicating the failure of BCDX2 to bind linear duplex DNA. ( D ) Juxtaposition of BCDX2 complex and RAD51 on gapped circular DNA. The DNA was preincubated with BCDX2 complex (90 nM) for 5 min and then supplemented with RAD51 (0.3 μM). After 5 min at 37°C, the products were visualized by electron microscopy. The white arrow indicates a RAD51 filament, and the black arrow indicates the BCDX2–DNA complexes.

    Article Snippet: [ ] Petukhova G, Van Komen S, Vergano S, Klein H, Sung P. Yeast Rad54 promotes Rad51-dependent homologous DNA pairing via ATP hydrolysis-driven change in DNA double helix conformation.

    Techniques: Electron Microscopy

    A. Schematic diagram showing DnaI-mediated loading of the G. kaustophilus ring helicase, DnaC. Monomers of DnaI (green) upon binding to ATP assemble into a hexameric ring around single-stranded DNA. The N-terminal tier of the DnaI ring interacts with the C-terminal tier of the helicase (brown) recruiting and opening up the helicase ring. Single-stranded DNA passes into the central channel of the ring. Binding of the DNA to the internal surface of the central channel stimulates the helicase activity and induces dissociation of DnaI ( Tsai et al ., 2009 ). B. Loading of helicase-primase bifunctional proteins. A helicase-primase protein initially interacts with the single-stranded DNA via its primase domains inducing an opening of the helicase ring with the DNA passing through the gap into the central channel. Binding of the DNA in the central cavity induces ring closure and activates the helicase-primase protein. The T7 gp4 primase-helicase crystallized as a heptamer (the seven subunits are coloured differently in the side view of the structure shown) but the translocating species along single-stranded DNA is believed to be the hexamer ( Toth et al ., 2003 ).

    Journal: Molecular Microbiology

    Article Title: Loading mechanisms of ring helicases at replication origins

    doi: 10.1111/j.1365-2958.2012.08012.x

    Figure Lengend Snippet: A. Schematic diagram showing DnaI-mediated loading of the G. kaustophilus ring helicase, DnaC. Monomers of DnaI (green) upon binding to ATP assemble into a hexameric ring around single-stranded DNA. The N-terminal tier of the DnaI ring interacts with the C-terminal tier of the helicase (brown) recruiting and opening up the helicase ring. Single-stranded DNA passes into the central channel of the ring. Binding of the DNA to the internal surface of the central channel stimulates the helicase activity and induces dissociation of DnaI ( Tsai et al ., 2009 ). B. Loading of helicase-primase bifunctional proteins. A helicase-primase protein initially interacts with the single-stranded DNA via its primase domains inducing an opening of the helicase ring with the DNA passing through the gap into the central channel. Binding of the DNA in the central cavity induces ring closure and activates the helicase-primase protein. The T7 gp4 primase-helicase crystallized as a heptamer (the seven subunits are coloured differently in the side view of the structure shown) but the translocating species along single-stranded DNA is believed to be the hexamer ( Toth et al ., 2003 ).

    Article Snippet: Structural distortions imposed on the DNA double helix and a squeeze-pump ATP-driven action of the two head-to-head hexameric helicases pulling the DNA in opposite directions force melting of the double helix (reviewed in ).

    Techniques: Binding Assay, Activity Assay

    A. Self-loading of the SV40 LTag DNA helicase. Two monomers of LTag bind head to head to the SV40 core ori sequence via their OBDs (green). One hexamer assembles around the double-stranded DNA followed by the second hexamer in a cooperative manner. The J-OBD cap is sandwiched between the helicase rings and forms a network of interactions holding the dodecamer together. The double-stranded DNA is melted by the reverse ‘iris pumping’ action of the two helicases and the single strands loop out of the dodecamer from side channels at the periphery of the helicase rings. Six positively charged side channels that could potentially provide outlets for the unwound single strands can be seen in the crystal structure of the SV40 LTag helicase domains ( Li et al ., 2003 ) and two of them, in the pink and yellow monomers, are highlighted in two different side views (60° rotated around the vertical axis relative to each other) of the hexameric ring. B. Self-loading mechanism of the papilloma virus E1 DNA helicase. Two E1-ATP subunits bind cooperatively to the specific ori sequence via their DBDs (green) to form two head-to-head mini-filaments. Upon ATP hydrolysis a double hexamer forms. Each hexamer encircles opposite single strands and the DBDs form a cap sandwiched between the two helicase hexamers. The two hexamers translocate 3′–5′ in the opposite directions along their respective strands, as shown by the opposing vertical arrows, effectively pumping the double-stranded DNA towards the centre of the complex. Two ‘pumping mechanisms’ are possible. The two hexameric rings dissociate and keep on moving in opposite directions expanding the ensuing ‘bubble’ of the separated DNA duplex. Alternatively, the two hexameric rings as they cross each other adhere to each other via interactions of their helicase domains resulting in a double hexamer that keeps on pumping the double-stranded DNA towards the centre of the complex yielding a ‘rabbit ear structure’. The latter is supported by the crystal structure of the E1 helicase domains showing two hexameric rings associated with each other but not collinear as each encircles a different single strand ( Enemark and Joshua-Tor, 2006 ). C. Loading of the MCM2–7 eukaryotic DNA helicase. MCM2–7 is loaded onto double-stranded DNA as a dodecamer, by the ORC/Cdc6 complex. Double-stranded DNA passes to the interior of the MCM2–7 ring channel through an opening between the MCM2 and 5 subunits. Binding of the dodecamer to double-stranded DNA distorts the DNA and additional pumping of the DNA by the two hexamers towards the centre of the dodecamer causes melting of the duplex inside the central cavity. Binding of the GINS/Cdc45 complex (green) to the periphery of the rings stabilizes open active forms of the rings, excluding different single strands from the central channels thus conferring 3′–5′ directionality along anti-parallel strands. The two hexamers then dissociate from each other and translocate in opposite directions.

    Journal: Molecular Microbiology

    Article Title: Loading mechanisms of ring helicases at replication origins

    doi: 10.1111/j.1365-2958.2012.08012.x

    Figure Lengend Snippet: A. Self-loading of the SV40 LTag DNA helicase. Two monomers of LTag bind head to head to the SV40 core ori sequence via their OBDs (green). One hexamer assembles around the double-stranded DNA followed by the second hexamer in a cooperative manner. The J-OBD cap is sandwiched between the helicase rings and forms a network of interactions holding the dodecamer together. The double-stranded DNA is melted by the reverse ‘iris pumping’ action of the two helicases and the single strands loop out of the dodecamer from side channels at the periphery of the helicase rings. Six positively charged side channels that could potentially provide outlets for the unwound single strands can be seen in the crystal structure of the SV40 LTag helicase domains ( Li et al ., 2003 ) and two of them, in the pink and yellow monomers, are highlighted in two different side views (60° rotated around the vertical axis relative to each other) of the hexameric ring. B. Self-loading mechanism of the papilloma virus E1 DNA helicase. Two E1-ATP subunits bind cooperatively to the specific ori sequence via their DBDs (green) to form two head-to-head mini-filaments. Upon ATP hydrolysis a double hexamer forms. Each hexamer encircles opposite single strands and the DBDs form a cap sandwiched between the two helicase hexamers. The two hexamers translocate 3′–5′ in the opposite directions along their respective strands, as shown by the opposing vertical arrows, effectively pumping the double-stranded DNA towards the centre of the complex. Two ‘pumping mechanisms’ are possible. The two hexameric rings dissociate and keep on moving in opposite directions expanding the ensuing ‘bubble’ of the separated DNA duplex. Alternatively, the two hexameric rings as they cross each other adhere to each other via interactions of their helicase domains resulting in a double hexamer that keeps on pumping the double-stranded DNA towards the centre of the complex yielding a ‘rabbit ear structure’. The latter is supported by the crystal structure of the E1 helicase domains showing two hexameric rings associated with each other but not collinear as each encircles a different single strand ( Enemark and Joshua-Tor, 2006 ). C. Loading of the MCM2–7 eukaryotic DNA helicase. MCM2–7 is loaded onto double-stranded DNA as a dodecamer, by the ORC/Cdc6 complex. Double-stranded DNA passes to the interior of the MCM2–7 ring channel through an opening between the MCM2 and 5 subunits. Binding of the dodecamer to double-stranded DNA distorts the DNA and additional pumping of the DNA by the two hexamers towards the centre of the dodecamer causes melting of the duplex inside the central cavity. Binding of the GINS/Cdc45 complex (green) to the periphery of the rings stabilizes open active forms of the rings, excluding different single strands from the central channels thus conferring 3′–5′ directionality along anti-parallel strands. The two hexamers then dissociate from each other and translocate in opposite directions.

    Article Snippet: Structural distortions imposed on the DNA double helix and a squeeze-pump ATP-driven action of the two head-to-head hexameric helicases pulling the DNA in opposite directions force melting of the double helix (reviewed in ).

    Techniques: Sequencing, Binding Assay

    A. The domain organization of DnaA. Structures of the N-terminal helicase-interacting domain (domain I) are shown in cyan, domain III comprising the AAA+ subdomain IIIA that binds ATP and the lid subdomain IIIB in yellow, and the C-terminal domain IV that interacts with double-stranded DNA in pink. The flexible linker domain II is missing while the ATP and Mg 2+ (grey ball) are shown in the active site of the AAA+ domain III. B. DnaA oligomerization. Two monomers of domain III are shown in yellow and grey with their arginines from Box VII and sensor II coloured in blue (yellow subunit) and red (grey subunit). The sensor II (red) of the grey monomer interacts in cis with the ATP (cyan) bound in its active site whereas the Box VII (blue) of the yellow monomer interacts in trans with the same ATP, as shown by the arrow. The ATP bound in the active site of the yellow monomer is shown with the relevant sensor II arginine defining the ATP-end of a filament, while the position of the Box VII of the grey monomer defines the R-end of the filament. C. The DnaA-ssDNA filament. A mini filament comprising four DnaA subunits bound to single-stranded DNA (red) and ATP (blue). The contiguous network of the α3/α4 and α5/α6 helices (domains III + IV) forming the single-stranded DNA binding surface lining the inside of the filament is highlighted in black ( Duderstadt et al ., 2011 ).

    Journal: Molecular Microbiology

    Article Title: Loading mechanisms of ring helicases at replication origins

    doi: 10.1111/j.1365-2958.2012.08012.x

    Figure Lengend Snippet: A. The domain organization of DnaA. Structures of the N-terminal helicase-interacting domain (domain I) are shown in cyan, domain III comprising the AAA+ subdomain IIIA that binds ATP and the lid subdomain IIIB in yellow, and the C-terminal domain IV that interacts with double-stranded DNA in pink. The flexible linker domain II is missing while the ATP and Mg 2+ (grey ball) are shown in the active site of the AAA+ domain III. B. DnaA oligomerization. Two monomers of domain III are shown in yellow and grey with their arginines from Box VII and sensor II coloured in blue (yellow subunit) and red (grey subunit). The sensor II (red) of the grey monomer interacts in cis with the ATP (cyan) bound in its active site whereas the Box VII (blue) of the yellow monomer interacts in trans with the same ATP, as shown by the arrow. The ATP bound in the active site of the yellow monomer is shown with the relevant sensor II arginine defining the ATP-end of a filament, while the position of the Box VII of the grey monomer defines the R-end of the filament. C. The DnaA-ssDNA filament. A mini filament comprising four DnaA subunits bound to single-stranded DNA (red) and ATP (blue). The contiguous network of the α3/α4 and α5/α6 helices (domains III + IV) forming the single-stranded DNA binding surface lining the inside of the filament is highlighted in black ( Duderstadt et al ., 2011 ).

    Article Snippet: Structural distortions imposed on the DNA double helix and a squeeze-pump ATP-driven action of the two head-to-head hexameric helicases pulling the DNA in opposite directions force melting of the double helix (reviewed in ).

    Techniques: Binding Assay

    A. Helicase loader-mediated loading in A. aeolicus . A schematic diagram showing directional helicase loader-mediated and DnaA-mediated loading mechanisms on the 5′ and 3′ strands, respectively, based upon the A. aeolicus system ( Duderstadt et al ., 2011 ). The DnaA filament is shown to wrap double-stranded DNA around the outside with flexible helicase-interacting domains (domain I) projecting out from the filament. The filament extends into the 5′ strand of the melted DUE but in this case with the ssDNA in the interior of the filament. The helicase loader (green) forms a continuous heterofilament with the ATP-end of the DnaA-ssDNA filament, by docking the arginine from its Box VII into the ATP binding site of the DnaA, and delivers the helicase (brown) in the correct orientation onto the 5′ strand. The interactions of the flexible N-terminal DnaA domains with the helicase deliver it onto the 3′ strand in the opposite direction. A side-view of the Bacillus stearothermophilus DnaB helicase is shown with the characteristic two-tier (N-terminal and C-terminal tiers) ring structure of bacterial helicases ( Bailey et al ., 2007 ). B. Organization of the E. coli oriC . The relative positions of DnaA binding sites (R, I and τ sites), NAP ( N ucleoid A ssociated P roteins) binding sites for IHF and FIS and the DUE are indicated. Binding of IHF between the R1 and R5 sites sharply bends the DNA, as indicated in the inset showing the crystal structure of IHF binding and bending double-stranded DNA. C. DnaA-mediated helicase loading in E. coli . A schematic model showing how two ring helicases are directionally loaded onto the E. coli oriC ( Ozaki and Katayama, 2012 ). The DUE is cooperatively melted via binding of DnaA and IHF and the first helicase is loaded onto the bottom (A-rich) strand directionally. Subsequent translocation forward (in the 5′–3′ direction) melts a larger segment of the duplex allowing loading of the second helicase in the top (T-rich) strand and in the opposite direction. In both cases loading is mediated by the flexible N-terminal helicase-interacting domains of DnaA projecting out of the filament. For the sake of simplicity no helicase loader is depicted but it may participate indirectly in the process by binding onto the C-terminal tier of the helicase ring forcing opening of the ring for loading to proceed.

    Journal: Molecular Microbiology

    Article Title: Loading mechanisms of ring helicases at replication origins

    doi: 10.1111/j.1365-2958.2012.08012.x

    Figure Lengend Snippet: A. Helicase loader-mediated loading in A. aeolicus . A schematic diagram showing directional helicase loader-mediated and DnaA-mediated loading mechanisms on the 5′ and 3′ strands, respectively, based upon the A. aeolicus system ( Duderstadt et al ., 2011 ). The DnaA filament is shown to wrap double-stranded DNA around the outside with flexible helicase-interacting domains (domain I) projecting out from the filament. The filament extends into the 5′ strand of the melted DUE but in this case with the ssDNA in the interior of the filament. The helicase loader (green) forms a continuous heterofilament with the ATP-end of the DnaA-ssDNA filament, by docking the arginine from its Box VII into the ATP binding site of the DnaA, and delivers the helicase (brown) in the correct orientation onto the 5′ strand. The interactions of the flexible N-terminal DnaA domains with the helicase deliver it onto the 3′ strand in the opposite direction. A side-view of the Bacillus stearothermophilus DnaB helicase is shown with the characteristic two-tier (N-terminal and C-terminal tiers) ring structure of bacterial helicases ( Bailey et al ., 2007 ). B. Organization of the E. coli oriC . The relative positions of DnaA binding sites (R, I and τ sites), NAP ( N ucleoid A ssociated P roteins) binding sites for IHF and FIS and the DUE are indicated. Binding of IHF between the R1 and R5 sites sharply bends the DNA, as indicated in the inset showing the crystal structure of IHF binding and bending double-stranded DNA. C. DnaA-mediated helicase loading in E. coli . A schematic model showing how two ring helicases are directionally loaded onto the E. coli oriC ( Ozaki and Katayama, 2012 ). The DUE is cooperatively melted via binding of DnaA and IHF and the first helicase is loaded onto the bottom (A-rich) strand directionally. Subsequent translocation forward (in the 5′–3′ direction) melts a larger segment of the duplex allowing loading of the second helicase in the top (T-rich) strand and in the opposite direction. In both cases loading is mediated by the flexible N-terminal helicase-interacting domains of DnaA projecting out of the filament. For the sake of simplicity no helicase loader is depicted but it may participate indirectly in the process by binding onto the C-terminal tier of the helicase ring forcing opening of the ring for loading to proceed.

    Article Snippet: Structural distortions imposed on the DNA double helix and a squeeze-pump ATP-driven action of the two head-to-head hexameric helicases pulling the DNA in opposite directions force melting of the double helix (reviewed in ).

    Techniques: Binding Assay, Immunohistofluorescence, Translocation Assay

    Alternation of DNA binding and looping function by varying salt concentration. ( A ) A 13 kb DNA with ΔLk = +14 was first compacted by 10 nM SMC1/3 under 0.5 pN force (0–800 s); DNA torsional response was tested by scanning the DNA extension from ΔLk = +30 to −30 with protein in solution (800–1300 s). Then we washed out the free protein in solution with protein free buffer containing 0.5 M NaCl; a fast DNA decompaction was observed (black arrow), and DNA torsional response was recovered to appear as naked DNA (1300–1750 s). Finally, we flush the flow cell with reaction buffer containing 0.1 M NaCl, and rapid DNA compaction was observed (gray arrow); DNA torsional response was also altered (1750–2150 s). ( B ) Average normalized DNA extension (extension relative to naked DNA contour length) at different salt concentration. First bar represents naked DNA before protein addition. The second bar represents the final DNA length in the presence of SMC1/3, normalized to naked DNA length; the average is 0.30 ± 0.03. After washing with reaction buffer containing 0.5M NaCl, the average DNA length is 0.92 ± 0.02 (third bar). After changing buffer back to reaction buffer containing 0.1 M NaCl, the average DNA normalized extension decreased to 0.50 ± 0.03 (fourth bar). Averages were computed over five experimental trials.

    Journal: Nucleic Acids Research

    Article Title: The SMC1-SMC3 cohesin heterodimer structures DNA through supercoiling-dependent loop formation

    doi: 10.1093/nar/gkt303

    Figure Lengend Snippet: Alternation of DNA binding and looping function by varying salt concentration. ( A ) A 13 kb DNA with ΔLk = +14 was first compacted by 10 nM SMC1/3 under 0.5 pN force (0–800 s); DNA torsional response was tested by scanning the DNA extension from ΔLk = +30 to −30 with protein in solution (800–1300 s). Then we washed out the free protein in solution with protein free buffer containing 0.5 M NaCl; a fast DNA decompaction was observed (black arrow), and DNA torsional response was recovered to appear as naked DNA (1300–1750 s). Finally, we flush the flow cell with reaction buffer containing 0.1 M NaCl, and rapid DNA compaction was observed (gray arrow); DNA torsional response was also altered (1750–2150 s). ( B ) Average normalized DNA extension (extension relative to naked DNA contour length) at different salt concentration. First bar represents naked DNA before protein addition. The second bar represents the final DNA length in the presence of SMC1/3, normalized to naked DNA length; the average is 0.30 ± 0.03. After washing with reaction buffer containing 0.5M NaCl, the average DNA length is 0.92 ± 0.02 (third bar). After changing buffer back to reaction buffer containing 0.1 M NaCl, the average DNA normalized extension decreased to 0.50 ± 0.03 (fourth bar). Averages were computed over five experimental trials.

    Article Snippet: We conclude that the SMC1-SMC3 heterodimer is able to restructure the DNA double helix into a series of loops, with a preference for positive writhe.

    Techniques: Binding Assay, Concentration Assay, Flow Cytometry

    DNA compaction by SMC1/3 mutants. DNA folding probability was plotted for hinge domain alone, hinge replacement mutant and headless mutant at 80 nM SMC1/3 protein concentration on ΔLk = +12 DNA ( A ) or nicked DNA ( B ). The number of experiments done was indicated on each bar. Error bars represent standard error.

    Journal: Nucleic Acids Research

    Article Title: The SMC1-SMC3 cohesin heterodimer structures DNA through supercoiling-dependent loop formation

    doi: 10.1093/nar/gkt303

    Figure Lengend Snippet: DNA compaction by SMC1/3 mutants. DNA folding probability was plotted for hinge domain alone, hinge replacement mutant and headless mutant at 80 nM SMC1/3 protein concentration on ΔLk = +12 DNA ( A ) or nicked DNA ( B ). The number of experiments done was indicated on each bar. Error bars represent standard error.

    Article Snippet: We conclude that the SMC1-SMC3 heterodimer is able to restructure the DNA double helix into a series of loops, with a preference for positive writhe.

    Techniques: Mutagenesis, Protein Concentration

    Changing DNA linking number results in SMC1/3 redistribution. Naked DNA at constant force shows a reversible extension versus linking number response centered at ΔLk = 0 (gray squares). However, this extension versus linking number response was altered in the presence of SMC1/3 protein (black squares). ( A and B ) experiments with DNA of ΔLk = +12 before protein addition, after reaction reaches equilibrium, DNA extension was measured as a function of ΔLk either from +30 to −30 (A), or from −30 to +30 (B). ( C and D ) Experiments with DNA of ΔLk = −15 before protein addition, after reaction reaches equilibrium, DNA extension was measured as a function of ΔLk either from +30 to −30 (C), or from −30 to +30 (D). Analysis of these repeated experiments in each group results in average peak linking number of ΔLk = 6.8 ± 1.2 ( n = 8), −10.5 ± 2.9 ( n = 3), 8 ± 1.5 ( n = 3) and −11.9 ± 1.2 ( n = 7) turn for A, B, C and D, respectively.

    Journal: Nucleic Acids Research

    Article Title: The SMC1-SMC3 cohesin heterodimer structures DNA through supercoiling-dependent loop formation

    doi: 10.1093/nar/gkt303

    Figure Lengend Snippet: Changing DNA linking number results in SMC1/3 redistribution. Naked DNA at constant force shows a reversible extension versus linking number response centered at ΔLk = 0 (gray squares). However, this extension versus linking number response was altered in the presence of SMC1/3 protein (black squares). ( A and B ) experiments with DNA of ΔLk = +12 before protein addition, after reaction reaches equilibrium, DNA extension was measured as a function of ΔLk either from +30 to −30 (A), or from −30 to +30 (B). ( C and D ) Experiments with DNA of ΔLk = −15 before protein addition, after reaction reaches equilibrium, DNA extension was measured as a function of ΔLk either from +30 to −30 (C), or from −30 to +30 (D). Analysis of these repeated experiments in each group results in average peak linking number of ΔLk = 6.8 ± 1.2 ( n = 8), −10.5 ± 2.9 ( n = 3), 8 ± 1.5 ( n = 3) and −11.9 ± 1.2 ( n = 7) turn for A, B, C and D, respectively.

    Article Snippet: We conclude that the SMC1-SMC3 heterodimer is able to restructure the DNA double helix into a series of loops, with a preference for positive writhe.

    Techniques:

    SMC1/3 concentration dependence of the compaction fraction and reaction rate for ΔLk = +12 DNA molecules. The equilibrium compaction fraction (black squares) was fit into Hill equation {θ = [SMC1/3] n /([K a ] n + [SMC1/3] n )} with a Hill coefficient n = 0.97 ± 0.16 and a K a = 1.2 ± 0.5 nM (black curve). The reaction rates (gray circles) were also fit into a Hill equation, with a Hill coefficient n r =1.20 ± 0.24, K r = 12 ± 3.9 nM (gray curve).

    Journal: Nucleic Acids Research

    Article Title: The SMC1-SMC3 cohesin heterodimer structures DNA through supercoiling-dependent loop formation

    doi: 10.1093/nar/gkt303

    Figure Lengend Snippet: SMC1/3 concentration dependence of the compaction fraction and reaction rate for ΔLk = +12 DNA molecules. The equilibrium compaction fraction (black squares) was fit into Hill equation {θ = [SMC1/3] n /([K a ] n + [SMC1/3] n )} with a Hill coefficient n = 0.97 ± 0.16 and a K a = 1.2 ± 0.5 nM (black curve). The reaction rates (gray circles) were also fit into a Hill equation, with a Hill coefficient n r =1.20 ± 0.24, K r = 12 ± 3.9 nM (gray curve).

    Article Snippet: We conclude that the SMC1-SMC3 heterodimer is able to restructure the DNA double helix into a series of loops, with a preference for positive writhe.

    Techniques: Concentration Assay

    Supercoiling dependence of DNA folding reaction by cohesin SMC1/3. ( A ) Total compaction rate. Positively supercoiled and nicked DNAs had the fastest rates of folding. Negatively supercoiled DNA was compacted more slowly, while un-nicked DNA with ΔLk = 0 was folded only very slowly. ( B ) Total compaction fraction showed a similar dependence on DNA topological state; note un-supercoiled un-nicked DNA was only slightly compacted. For each group of DNA, the number of experiments n = 15, 19, 15 and 15 for nicked DNA, ΔLk = −15, 0 and +12 DNA, respectively.

    Journal: Nucleic Acids Research

    Article Title: The SMC1-SMC3 cohesin heterodimer structures DNA through supercoiling-dependent loop formation

    doi: 10.1093/nar/gkt303

    Figure Lengend Snippet: Supercoiling dependence of DNA folding reaction by cohesin SMC1/3. ( A ) Total compaction rate. Positively supercoiled and nicked DNAs had the fastest rates of folding. Negatively supercoiled DNA was compacted more slowly, while un-nicked DNA with ΔLk = 0 was folded only very slowly. ( B ) Total compaction fraction showed a similar dependence on DNA topological state; note un-supercoiled un-nicked DNA was only slightly compacted. For each group of DNA, the number of experiments n = 15, 19, 15 and 15 for nicked DNA, ΔLk = −15, 0 and +12 DNA, respectively.

    Article Snippet: We conclude that the SMC1-SMC3 heterodimer is able to restructure the DNA double helix into a series of loops, with a preference for positive writhe.

    Techniques:

    Archaeal/eukaryotic helicase interacting with DNA. ( A ) DNA opening and unwinding by the Mcm2-7 helicase: the two motor domains that move along the DNA are at opposite ends of a DNA-loaded helicase double ring, with the N-terminal DNA-binding domains (NTD) in the middle. Froelich, Kang et al. propose that the motor domains push the duplex DNA towards the middle of the helicase, hence promoting melting of the DNA at the origin of replication and trapping of the leading strand template by the NTD. The rings then separate and travel in opposite directions, with one ring sliding along each of the leading strand templates from the two replication forks. ( B ) Crystal structure of the MCM helicase (orange) bound to single-stranded DNA (light blue). The DNA circles around the MCM central channel in a clockwise direction (when travelling from the 5′-end to 3′-end and viewing the ring from its C-terminal face).

    Journal: eLife

    Article Title: Preparing to unwind

    doi: 10.7554/eLife.02618

    Figure Lengend Snippet: Archaeal/eukaryotic helicase interacting with DNA. ( A ) DNA opening and unwinding by the Mcm2-7 helicase: the two motor domains that move along the DNA are at opposite ends of a DNA-loaded helicase double ring, with the N-terminal DNA-binding domains (NTD) in the middle. Froelich, Kang et al. propose that the motor domains push the duplex DNA towards the middle of the helicase, hence promoting melting of the DNA at the origin of replication and trapping of the leading strand template by the NTD. The rings then separate and travel in opposite directions, with one ring sliding along each of the leading strand templates from the two replication forks. ( B ) Crystal structure of the MCM helicase (orange) bound to single-stranded DNA (light blue). The DNA circles around the MCM central channel in a clockwise direction (when travelling from the 5′-end to 3′-end and viewing the ring from its C-terminal face).

    Article Snippet: To duplicate a molecule of double-stranded DNA within a cell, an ‘initiation factor’ recognises a stretch of DNA called an origin of replication and then recruits an enzyme called a helicase that goes on to unwind the double helix.

    Techniques: Binding Assay