scholarly journals Single-molecule visualization of human RECQ5 interactions with single-stranded DNA recombination intermediates

2020 ◽  
Author(s):  
Chaoyou Xue ◽  
Lucia Molnarova ◽  
Justin B Steinfeld ◽  
Weixing Zhao ◽  
Chujian Ma ◽  
...  
Keyword(s):  
Single Molecule ◽  
Atp Hydrolysis ◽  
Protein A ◽  
Dna Recombination ◽  
Binding Activity ◽  
Negative Regulator ◽  
Genome Maintenance ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Translocation Velocity

Abstract RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.

scholarly journals RADX condenses single-stranded DNA to antagonize RAD51 loading

2020 ◽  
Vol 48 (14) ◽  
pp. 7834-7843
Author(s):  
Hongshan Zhang ◽  
Jeffrey M Schaub ◽  
Ilya J Finkelstein
Keyword(s):  
Single Molecule ◽  
Protein A ◽  
Negative Regulator ◽  
Replication Forks ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
In Trans ◽  
Ssdna Binding Proteins ◽  
Stalled Replication Forks

Abstract RADX is a mammalian single-stranded DNA-binding protein that stabilizes telomeres and stalled replication forks. Cellular biology studies have shown that the balance between RADX and Replication Protein A (RPA) is critical for DNA replication integrity. RADX is also a negative regulator of RAD51-mediated homologous recombination at stalled forks. However, the mechanism of RADX acting on DNA and its interactions with RPA and RAD51 are enigmatic. Using single-molecule imaging of the key proteins in vitro, we reveal that RADX condenses ssDNA filaments, even when the ssDNA is coated with RPA at physiological protein ratios. RADX compacts RPA-coated ssDNA filaments via higher-order assemblies that can capture ssDNA in trans. Furthermore, RADX blocks RPA displacement by RAD51 and prevents RAD51 loading on ssDNA. Our results indicate that RADX is an ssDNA condensation protein that inhibits RAD51 filament formation and may antagonize other ssDNA-binding proteins on RPA-coated ssDNA.

scholarly journals RADX condenses single-stranded DNA to antagonize RAD51 loading

2020 ◽  
Author(s):  
Hongshan Zhang ◽  
Jeffrey M. Schaub ◽  
Ilya J. Finkelstein
Keyword(s):  
Protein A ◽  
Negative Regulator ◽  
Cellular Biology ◽  
Replication Forks ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
In Trans ◽  
Ssdna Binding Proteins ◽  
Stalled Replication Forks

AbstractRADX is a mammalian single-stranded DNA-binding protein that stabilizes telomeres and stalled replication forks. Cellular biology studies have shown that the balance between RADX and Replication Protein A (RPA) activities is critical for DNA replication integrity. RADX is also a negative regulator of RAD51-mediated homologous recombination at stalled forks. However, the mechanism of RADX acting on DNA and its interactions with RPA and RAD51 are enigmatic. Using singlemolecule imaging of the key proteins in vitro, we reveal that RADX condenses ssDNA filaments, even when the ssDNA is coated with RPA at physiological protein ratios. RADX compacts RPA-coated ssDNA filaments via higher-order assemblies that can capture ssDNA in trans. Furthermore, RADX blocks RPA displacement by RAD51 and prevents RAD51 loading on ssDNA. Our results indicate that RADX is an ssDNA condensation protein that inhibits RAD51 filament formation and may antagonize other ssDNA-binding proteins on RPA-coated ssDNA.

scholarly journals Human replication protein A binds single-stranded DNA in two distinct complexes.

1994 ◽  
Vol 14 (6) ◽  
pp. 3993-4001 ◽  
Author(s):  
L J Blackwell ◽  
J A Borowiec
Keyword(s):  
Protein A ◽  
Dna Recombination ◽  
Replication Protein A ◽  
Replication Protein ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Ssdna Binding Protein ◽  
Eukaryotic Dna ◽  
Gel Shift

Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.

Human replication protein A binds single-stranded DNA in two distinct complexes

1994 ◽  
Vol 14 (6) ◽  
pp. 3993-4001
Author(s):  
L J Blackwell ◽  
J A Borowiec
Keyword(s):  
Protein A ◽  
Dna Recombination ◽  
Replication Protein A ◽  
Replication Protein ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Ssdna Binding Protein ◽  
Eukaryotic Dna ◽  
Gel Shift

Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8-10nt complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30nt complex), a complex similar to that described by Kim et al. (C. Kim, R. O. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8-10nt and hRPA30nt complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.

scholarly journals Single-molecule visualization of human BLM helicase as it acts upon double- and single-stranded DNA substrates

2019 ◽  
Vol 47 (21) ◽  
pp. 11225-11237 ◽  
Author(s):  
Chaoyou Xue ◽  
James M Daley ◽  
Xiaoyu Xue ◽  
Justin Steinfeld ◽  
Youngho Kwon ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Genetic Disorder ◽  
Protein A ◽  
Base Pairs ◽  
Single Stranded Dna ◽  
Double Stranded Dna ◽  
Blm Helicase ◽  
Dna Replication And Repair ◽  
Regulatory Effects

Abstract Bloom helicase (BLM) and its orthologs are essential for the maintenance of genome integrity. BLM defects represent the underlying cause of Bloom Syndrome, a rare genetic disorder that is marked by strong cancer predisposition. BLM deficient cells accumulate extensive chromosomal aberrations stemming from dysfunctions in homologous recombination (HR). BLM participates in several HR stages and helps dismantle potentially harmful HR intermediates. However, much remains to be learned about the molecular mechanisms of these BLM-mediated regulatory effects. Here, we use DNA curtains to directly visualize the activity of BLM helicase on single molecules of DNA. Our data show that BLM is a robust helicase capable of rapidly (∼70–80 base pairs per second) unwinding extensive tracts (∼8–10 kilobases) of double-stranded DNA (dsDNA). Importantly, we find no evidence for BLM activity on single-stranded DNA (ssDNA) that is bound by replication protein A (RPA). Likewise, our results show that BLM can neither associate with nor translocate on ssDNA that is bound by the recombinase protein RAD51. Moreover, our data reveal that the presence of RAD51 also blocks BLM translocation on dsDNA substrates. We discuss our findings within the context of potential regulator roles for BLM helicase during DNA replication and repair.

scholarly journals Recruitment of Replication Protein A by the Papillomavirus E1 Protein and Modulation by Single-Stranded DNA

2004 ◽  
Vol 78 (4) ◽  
pp. 1605-1615 ◽  
Author(s):  
Yueh-Ming Loo ◽  
Thomas Melendy
Keyword(s):  
Dna Replication ◽  
Protein A ◽  
Replication Protein A ◽  
T Antigen ◽  
Replication Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Physical Interaction ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Papillomavirus Dna

ABSTRACT With the exception of viral proteins E1 and E2, papillomaviruses depend heavily on host replication machinery for replication of their viral genome. E1 and E2 are known to recruit many of the necessary cellular replication factors to the viral origin of replication. Previously, we reported a physical interaction between E1 and the major human single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA). E1 was determined to bind to the 70-kDa subunit of RPA, RPA70. In this study, using E1-affinity coprecipitation and enzyme-linked immunosorbent assay-based interaction assays, we show that E1 interacts with the major ssDNA-binding domain of RPA. Consistent with our previous report, no measurable interaction between E1 and the two smaller subunits of RPA was detected. The interaction of E1 with RPA was substantially inhibited by ssDNA. The extent of this inhibition was dependent on the length of the DNA. A 31-nucleotide (nt) oligonucleotide strongly inhibited the E1-RPA interaction, while a 16-nt oligonucleotide showed an intermediate level of inhibition. In contrast, a 10-nt oligonucleotide showed no observable effect on the E1-RPA interaction. This inhibition was not dependent on the sequence of the DNA. Furthermore, ssDNA also inhibited the interaction of RPA with papillomavirus E2, simian virus 40 T antigen, human polymerase alpha-primase, and p53. Taken together, our results suggest a potential role for ssDNA in modulating RPA-protein interactions, in particular, the RPA-E1 interactions during papillomavirus DNA replication. A model for recruitment of RPA by E1 during papillomavirus DNA replication is proposed.

scholarly journals Structural insights into the unique single-stranded DNA-binding mode of Helicobacter pylori DprA

2013 ◽  
Vol 42 (5) ◽  
pp. 3478-3491 ◽  
Author(s):  
Wei Wang ◽  
Jingjin Ding ◽  
Ying Zhang ◽  
Yonglin Hu ◽  
Da-Cheng Wang
Keyword(s):  
Helicobacter Pylori ◽  
Bacterial Species ◽  
Complex Structure ◽  
Protein A ◽  
Binding Mode ◽  
Binding Assays ◽  
Mobility Shift ◽  
Single Stranded Dna ◽  
Exogenous Dna ◽  
Ssdna Binding

Abstract Natural transformation (NT) in bacteria is a complex process, including binding, uptake, transport and recombination of exogenous DNA into the chromosome, consequently generating genetic diversity and driving evolution. DNA processing protein A (DprA), which is distributed among virtually all bacterial species, is involved in binding to the internalized single-stranded DNA (ssDNA) and promoting the loading of RecA on ssDNA during NTs. Here we present the structures of DNA_processg_A (DprA) domain of the Helicobacter pylori DprA (HpDprA) and its complex with an ssDNA at 2.20 and 1.80 Å resolutions, respectively. The complex structure revealed for the first time how the conserved DprA domain binds to ssDNA. Based on structural comparisons and binding assays, a unique ssDNA-binding mode is proposed: the dimer of HpDprA binds to ssDNA through two small, positively charged binding pockets of the DprA domains with classical Rossmann folds and the key residue Arg52 is re-oriented to ‘open’ the pocket in order to accommodate one of the bases of ssDNA, thus enabling HpDprA to grasp substrate with high affinity. This mode is consistent with the oligomeric composition of the complex as shown by electrophoretic mobility-shift assays and static light scattering measurements, but differs from the direct polymeric complex of Streptococcus pneumoniae DprA–ssDNA.

scholarly journals Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Thomas J Etheridge ◽  
Desiree Villahermosa ◽  
Eduard Campillo-Funollet ◽  
Alex David Herbert ◽  
Anja Irmisch ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Single Molecule ◽  
Chromosomal Rearrangements ◽  
Binding Activity ◽  
Single Molecule Tracking ◽  
Ssdna Binding ◽  
Dna Binding Activity ◽  
Gross Chromosomal Rearrangements ◽  
Genes Encoding

The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1. Using defined mutants in genes encoding the core Smc5/6 complex subunits, we show that the Nse3 double-stranded DNA binding activity and the arginine fingers of the two Smc5/6 ATPase binding sites are critical for chromatin association. Interestingly, disrupting the single-stranded DNA (ssDNA) binding activity at the hinge region does not prevent chromatin association but leads to elevated levels of gross chromosomal rearrangements during replication restart. This is consistent with a downstream function for ssDNA binding in regulating homologous recombination.

scholarly journals Human HELB is a processive motor protein which catalyses RPA clearance from single-stranded DNA

2021 ◽  
Author(s):  
Silvia Hormeno ◽  
Oliver J Wilkinson ◽  
Clara Aicart-Ramos ◽  
Sahiti Kuppa ◽  
Edwin Antony ◽  
...  
Keyword(s):  
Dna Replication ◽  
Direct Observation ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Motor Protein ◽  
Dna Unwinding ◽  
Helicase Activity ◽  
Dna Loops ◽  
Single Stranded Dna ◽  
A Site

Human HELB is a poorly-characterised helicase suggested to play both positive and negative regulatory roles in DNA replication and recombination. In this work, we used bulk and single molecule approaches to characterise the biochemical activities of HELB protein with a particular focus on its interactions with RPA and RPA-ssDNA filaments. HELB is a monomeric protein which binds tightly to ssDNA with a site size of ~20 nucleotides. It couples ATP hydrolysis to translocation along ssDNA in the 5′-to-3′ direction accompanied by the formation of DNA loops and with an efficiency of 1 ATP per base. HELB also displays classical helicase activity but this is very weak in the absence of an assisting force. HELB binds specifically to human RPA which enhances its ATPase and ssDNA translocase activities but inhibits DNA unwinding. Direct observation of HELB on RPA nucleoprotein filaments shows that translocating HELB concomitantly clears RPA from single-stranded DNA.

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