Mechanism of RPA-Facilitated Processive DNA Unwinding by the Eukaryotic CMG Helicase

AbstractThe DNA double helix is unwound by the Cdc45/Mcm2-7/GINS (CMG) complex at the eukaryotic replication fork. While isolated CMG unwinds duplex DNA very slowly, its fork unwinding rate is stimulated by an order of magnitude by single-stranded DNA binding protein, RPA. However, the molecular mechanism by which RPA enhances CMG helicase activity remained elusive. Here, we demonstrate that engagement of CMG with parental double-stranded DNA (dsDNA) at the replication fork impairs its helicase activity, explaining the slow DNA unwinding by isolated CMG. Using single-molecule and ensemble biochemistry, we show that binding of RPA to the excluded DNA strand prevents duplex engagement by the helicase and speeds up CMG-mediated DNA unwinding. When stalled due to dsDNA interaction, DNA rezipping-induced helicase backtracking re-establishes productive helicase-fork engagement underscoring the significance of plasticity in helicase action. Together, our results elucidate the dynamics of CMG at the replication fork and reveal how other replisome components can mediate proper DNA engagement by the replicative helicase to achieve efficient fork progression.

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The Sulfolobus solfataricus GINS Complex Stimulates DNA Binding and Processive DNA Unwinding by Minichromosome Maintenance Helicase

Journal of Bacteriology ◽

10.1128/jb.00496-15 ◽

2015 ◽

Vol 197 (21) ◽

pp. 3409-3420 ◽

Cited By ~ 12

Author(s):

Shiwei Lang ◽

Li Huang

Keyword(s):

Dna Binding ◽

Atpase Activity ◽

Replication Fork ◽

Molecular Motor ◽

Sulfolobus Solfataricus ◽

Dna Unwinding ◽

Helicase Activity ◽

Minichromosome Maintenance ◽

Content Type ◽

Double Stranded Dna

ABSTRACTGINS is a key component of the eukaryotic Cdc45-minichromosome maintenance (MCM)-GINS (CMG) complex, which unwinds duplex DNA at the moving replication fork. Archaeal GINS complexes have been shown to stimulate the helicase activity of their cognate MCM mainly by elevating its ATPase activity. Here, we report that GINS from the thermoacidophilic crenarchaeonSulfolobus solfataricus(SsoGINS) is capable of DNA binding and binds preferentially to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA). Notably, SsoGINS binds more strongly to dsDNA with a 5′ ssDNA tail than to dsDNA with a 3′ tail and more strongly to an ssDNA fragment blocked at the 3′ end than to one at the 5′ end with a biotin-streptavidin (SA) complex, suggesting the ability of the protein complex to slide in a 5′-to-3′ direction along ssDNA. DNA-bound SsoGINS enhances DNA binding by SsoMCM. Furthermore, SsoGINS increases the helicase activity of SsoMCM. However, the ATPase activity of SsoMCM is not affected by SsoGINS. Our results suggest that SsoGINS facilitates processive DNA unwinding by SsoMCM by enhancing the binding of the helicase to DNA. We propose that SsoGINS stabilizes the interaction of SsoMCM with the replication fork and moves along with the helicase as the fork progresses.IMPORTANCEGINS is a key component of the eukaryotic Cdc45-MCM-GINS complex, a molecular motor that drives the unwinding of DNA in front of the replication fork.Archaeaalso encode GINS, which interacts with MCM, the helicase. But how archaeal GINS serves its role remains to be understood. In this study, we show that GINS from the hyperthermophilic archaeonSulfolobus solfataricusis able to bind to DNA and slide along ssDNA in a 5′-to-3′ direction. Furthermore,SulfolobusGINS enhances DNA binding by MCM, which slides along ssDNA in a 3′-to-5′ direction. Taken together, these results suggest thatSulfolobusGINS may stabilize the interaction of MCM with the moving replication fork, facilitating processive DNA unwinding.

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Ionic strength modulates HU protein-induced DNA supercoiling

10.1101/2021.10.14.464438 ◽

2021 ◽

Author(s):

Alexander Zhang ◽

Yan Yan ◽

Fenfei Leng ◽

David Dunlap ◽

Laura Finzi

Keyword(s):

Single Molecule ◽

Structural Changes ◽

Magnetic Measurements ◽

Double Helix ◽

Bacterial Genome ◽

Dna Supercoiling ◽

Coli Strain ◽

Dna Unwinding ◽

Dna Compaction ◽

Dna Double Helix

The histone-like protein from E. coli strain U93 (HU) is an abundant nucleoid-associated protein that contributes to the compaction of the bacterial genome as well as to the regulation of many of its transactions. Despite many years of investigations, the way and extent to which HU binding alters the DNA double helix and/or generates hierarchical structures using DNA as a scaffold is not completely understood. Here we combined single-molecule magnetic measurements with circular dichroism studies to monitor structural changes in the DNA-HU fiber as HU concentration was increased from 0 to 1000 nM under low and physiological monovalent salt conditions. We confirmed that DNA compaction correlated with HU concentration in a biphasic manner but DNA unwinding varied monotonically with HU concentration in 100 mM KCl. Instead, in more physiological 200 mM salt conditions, DNA compaction was monotonic while HU-induced DNA unwinding was negligible. Differential compaction and unwinding of DNA may be part of the response of bacteria to large variations in salt concentrations.

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Mercury Electrodes in Nucleic Acid Electrochemistry: Sensitive Analytical Tools and Probes of DNA Structure. A Review

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc20040715 ◽

2004 ◽

Vol 69 (4) ◽

pp. 715-747 ◽

Cited By ~ 40

Author(s):

Miroslav Fojta

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Double Helix ◽

Dna Structure ◽

Electrochemical Analysis ◽

Label Free ◽

Helix Formation ◽

Mercury Electrodes ◽

Dna Strand ◽

Dna Double Helix

This review is devoted to applications of mercury electrodes in the electrochemical analysis of nucleic acids and in studies of DNA structure and interactions. At the mercury electrodes, nucleic acids yield faradaic signals due to redox processes involving adenine, cytosine and guanine residues, and tensammetric signals due to adsorption/desorption of polynucleotide chains at the electrode surface. Some of these signals are highly sensitive to DNA structure, providing information about conformation changes of the DNA double helix, formation of DNA strand breaks as well as covalent or non-covalent DNA interactions with small molecules (including genotoxic agents, drugs, etc.). Measurements at mercury electrodes allow for determination of small quantities of unmodified or electrochemically labeled nucleic acids. DNA-modified mercury electrodes have been used as biodetectors for DNA damaging agents or as detection electrodes in DNA hybridization assays. Mercury film and solid amalgam electrodes possess similar features in the nucleic acid analysis to mercury drop electrodes. On the contrary, intrinsic (label-free) DNA electrochemical responses at other (non-mercury) solid electrodes cannot provide information about small changes of the DNA structure. A review with 188 references.

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Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1518028112 ◽

2015 ◽

Vol 112 (50) ◽

pp. E6852-E6861 ◽

Cited By ~ 23

Author(s):

Behzad Rad ◽

Anthony L. Forget ◽

Ronald J. Baskin ◽

Stephen C. Kowalczykowski

Keyword(s):

Single Molecule ◽

Fluorescent Sensor ◽

Holliday Junctions ◽

Dna Unwinding ◽

Recq Helicase ◽

Dna Breaks ◽

Dna Helicases ◽

Double Stranded Dna ◽

Functional Forms ◽

And Migration

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40–60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.

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Geometry of the DNA strands within the RecA nucleofilament: role in homologous recombination

Quarterly Reviews of Biophysics ◽

10.1017/s0033583504003956 ◽

2003 ◽

Vol 36 (4) ◽

pp. 429-453 ◽

Cited By ~ 29

Author(s):

Chantal Prévost ◽

Masayuki Takahashi

Keyword(s):

Homologous Recombination ◽

Structural Information ◽

Double Helix ◽

Genetic Material ◽

Strand Exchange ◽

Double Stranded Dna ◽

Dna Deformation ◽

Architectural Proteins ◽

Dna Strands ◽

Dna Strand

1. Introduction 4302. Transformations of the RecA filament 4312.1 The different forms of the RecA filament 4312.2 Orientation and position of the RecA monomers in the active filament 4332.3 Transmission of structural information along the filament 4333. RecA-induced DNA deformations 4353.1 Characteristics of RecA-bound DNA 4353.2 Stretching properties of double-stranded DNA 4363.3 DNA bound to architectural proteins 4373.4 Implications for RecA-induced DNA deformations 4383.5 Axial distribution of the DNA stretching deformation 4384. Contacts between RecA and the DNA strands 4404.1 The DNA-binding sites 4404.2 Possible arrangement of loops L1 and L2 and the three bound strands of DNA 4425. Strand arrangement during pairing reorganization 4445.1 Hypotheses for DNA strand association 4445.2 Association via major or minor grooves 4465.3 Post-strand exchange geometries 4466. Conclusion 4477. Acknowledgments 4488. References 448Homologous recombination consists of exchanging DNA strands of identical or almost identical sequence. This process is important for both DNA repair and DNA segregation. In prokaryotes, it involves the formation of long helical filaments of the RecA protein on DNA. These filaments incorporate double-stranded DNA from the cell's genetic material, recognize sequence homology and promote strand exchange between the two DNA segments. DNA processing by these nucleofilaments is characterized by large amplitude deformations of the double helix, which is stretched by 50% and unwound by 40% with respect to B-DNA. In this article, information concerning the structure and interactions of the RecA, DNA and ATP molecules involved in DNA strand exchange is gathered and analyzed to present a view of their possible arrangement within the filament, their behavior during strand exchange and during ATP hydrolysis, the mechanism of RecA-promoted DNA deformation and the role of DNA deformation in the process of homologous recombination. In particular, the unusual characteristics of DNA within the RecA filament are compared to the DNA deformations locally induced by architectural proteins which bind in the DNA minor groove. The possible role and location of two flexible loops of RecA are discussed.

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Direct Single-Molecule Observations of Local Denaturation of a DNA Double Helix under a Negative Supercoil State

Analytical Chemistry ◽

10.1021/acs.analchem.5b00044 ◽

2015 ◽

Vol 87 (6) ◽

pp. 3490-3497 ◽

Cited By ~ 4

Author(s):

Shunsuke Takahashi ◽

Shinya Motooka ◽

Tomohiro Usui ◽

Shohei Kawasaki ◽

Hidefumi Miyata ◽

...

Keyword(s):

Single Molecule ◽

Double Helix ◽

Dna Double Helix

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The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader

eLife ◽

10.7554/elife.64232 ◽

2021 ◽

Vol 10 ◽

Author(s):

Neha Puri ◽

Amy J Fernandez ◽

Valerie L O'Shea Murray ◽

Sarah McMillan ◽

James L Keck ◽

...

Keyword(s):

Replication Fork ◽

Substrate Recognition ◽

Dna Unwinding ◽

Replicative Helicase ◽

E Coli ◽

Helicase Loading ◽

Dnab Helicase ◽

Replication Restart ◽

Clamp Loading ◽

Aaa Atpases

In many bacteria and in eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+ ATPases. How loading factors use ATP to control helicase deposition is poorly understood. Here, we dissect how specific ATPase elements of E. coli DnaC, an archetypal loader for the bacterial DnaB helicase, play distinct roles in helicase loading and the activation of DNA unwinding. We identify a new element, the arginine-coupler, which regulates the switch-like behavior of DnaC to prevent futile ATPase cycling and maintains loader responsiveness to replication restart systems. Our data help explain how the ATPase cycle of a AAA+-family helicase loader is channeled into productive action on its target; comparative studies indicate elements analogous to the Arg-coupler are present in related, switch-like AAA+ proteins that control replicative helicase loading in eukaryotes, as well as polymerase clamp loading and certain classes of DNA transposases.

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

10.1101/2021.05.27.445972 ◽

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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A conserved Mcm4 motif is required for Mcm2-7 double-hexamer formation and origin DNA unwinding

eLife ◽

10.7554/elife.45538 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Kanokwan Champasa ◽

Caitlin Blank ◽

Larry J Friedman ◽

Jeff Gelles ◽

Stephen P Bell

Keyword(s):

Single Molecule ◽

Time Window ◽

Dna Melting ◽

Dna Unwinding ◽

Limited Time ◽

Wild Type ◽

Replicative Helicase ◽

Dynamic Mechanisms ◽

Origin Licensing ◽

Single Molecule Studies

Licensing of eukaryotic origins of replication requires DNA loading of two copies of the Mcm2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally. To understand the formation and importance of this double-hexamer, we identified mutations in a conserved and essential Mcm4 motif that permit loading of two Mcm2-7 complexes but are defective for double-hexamer formation. Single-molecule studies show mutant Mcm2-7 forms initial hexamer-hexamer interactions; however, the resulting complex is unstable. Kinetic analyses of wild-type and mutant Mcm2-7 reveal a limited time window for double-hexamer formation following second Mcm2-7 association, suggesting that this process is facilitated. Double-hexamer formation is required for extensive origin DNA unwinding but not initial DNA melting or recruitment of helicase-activation proteins (Cdc45, GINS, Mcm10). Our findings elucidate dynamic mechanisms of origin licensing, and identify the transition between initial DNA melting and extensive unwinding as the first initiation event requiring double-hexamer formation.

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