DNA Unwinding Is an MCM Complex-dependent and ATP Hydrolysis-dependent Process

Minichromosome maintenance proteins (Mcm) are essential in all eukaryotes and are absolutely required for initiation of DNA replication. The eukaryotic and archaeal Mcm proteins have conserved helicase motifs and exhibit DNA helicase and ATP hydrolysis activitiesin vitro. Although the Mcm proteins have been proposed to be the replicative helicase, the enzyme that melts the DNA helix at the replication fork, their function during cellular DNA replication elongation is still unclear. Using nucleoplasmic extract (NPE) fromXenopus laeviseggs and six purified polyclonal antibodies generated against each of theXenopusMcm proteins, we have demonstrated that Mcm proteins are required during DNA replication and DNA unwinding after initiation of replication. Quantitative depletion of Mcms from the NPE results in normal replication and unwinding, confirming that Mcms are required before pre-replicative complex assembly and dispensable thereafter. Replication and unwinding are inhibited when pooled neutralizing antibodies against the six different Mcm2–7 proteins are added during NPE incubation. Furthermore, replication is blocked by the addition of the Mcm antibodies after an initial period of replication in the NPE, visualized by a pulse of radiolabeled nucleotide at the same time as antibody addition. Addition of the cyclin-dependent kinase 2 inhibitor p21cip1specifically blocks origin firing but does not prevent helicase action. When p21cip1is added, followed by the non-hydrolyzable analog ATPγS to block helicase function, unwinding is inhibited, demonstrating that plasmid unwinding is specifically attributable to an ATP hydrolysis-dependent function. These data support the hypothesis that the Mcm protein complex functions as the replicative helicase.

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Ciprofloxacin is an inhibitor of the Mcm2-7 replicative helicase

Bioscience Reports ◽

10.1042/bsr20130083 ◽

2013 ◽

Vol 33 (5) ◽

Cited By ~ 21

Author(s):

Nicholas Simon ◽

Matthew L. Bochman ◽

Sandlin Seguin ◽

Jeffrey L. Brodsky ◽

William L. Seibel ◽

...

Keyword(s):

Dna Replication ◽

Dna Helicase ◽

Dna Unwinding ◽

Helicase Activity ◽

Minichromosome Maintenance ◽

Replicative Helicase ◽

Eukaryotic Dna ◽

Minichromosome Maintenance Protein ◽

Ciprofloxacin Resistance

Most currently available small molecule inhibitors of DNA replication lack enzymatic specificity, resulting in deleterious side effects during use in cancer chemotherapy and limited experimental usefulness as mechanistic tools to study DNA replication. Towards development of targeted replication inhibitors, we have focused on Mcm2-7 (minichromosome maintenance protein 2–7), a highly conserved helicase and key regulatory component of eukaryotic DNA replication. Unexpectedly we found that the fluoroquinolone antibiotic ciprofloxacin preferentially inhibits Mcm2-7. Ciprofloxacin blocks the DNA helicase activity of Mcm2-7 at concentrations that have little effect on other tested helicases and prevents the proliferation of both yeast and human cells at concentrations similar to those that inhibit DNA unwinding. Moreover, a previously characterized mcm mutant (mcm4chaos3) exhibits increased ciprofloxacin resistance. To identify more potent Mcm2-7 inhibitors, we screened molecules that are structurally related to ciprofloxacin and identified several that compromise the Mcm2-7 helicase activity at lower concentrations. Our results indicate that ciprofloxacin targets Mcm2-7 in vitro, and support the feasibility of developing specific quinolone-based inhibitors of Mcm2-7 for therapeutic and experimental applications.

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DisA Limits RecG Activities at Stalled or Reversed Replication Forks

Cells ◽

10.3390/cells10061357 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1357

Author(s):

Rubén Torres ◽

Carolina Gándara ◽

Begoña Carrasco ◽

Ignacio Baquedano ◽

Silvia Ayora ◽

...

Keyword(s):

Atp Hydrolysis ◽

Holliday Junctions ◽

Genome Integrity ◽

Stable Complex ◽

Dna Unwinding ◽

Replication Forks ◽

Dna Structures ◽

Checkpoint Protein

The DNA damage checkpoint protein DisA and the branch migration translocase RecG are implicated in the preservation of genome integrity in reviving haploid Bacillus subtilis spores. DisA synthesizes the essential cyclic 3′, 5′-diadenosine monophosphate (c‑di-AMP) second messenger and such synthesis is suppressed upon replication perturbation. In vitro, c-di-AMP synthesis is suppressed when DisA binds DNA structures that mimic stalled or reversed forks (gapped forks or Holliday junctions [HJ]). RecG, which does not form a stable complex with DisA, unwinds branched intermediates, and in the presence of a limiting ATP concentration and HJ DNA, it blocks DisA-mediated c-di-AMP synthesis. DisA pre-bound to a stalled or reversed fork limits RecG-mediated ATP hydrolysis and DNA unwinding, but not if RecG is pre-bound to stalled or reversed forks. We propose that RecG-mediated fork remodeling is a genuine in vivo activity, and that DisA, as a molecular switch, limits RecG-mediated fork reversal and fork restoration. DisA and RecG might provide more time to process perturbed forks, avoiding genome breakage.

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DNA and ATP Binding Activities of the Baculovirus DNA Helicase P143

Journal of Virology ◽

10.1128/jvi.75.15.7206-7209.2001 ◽

2001 ◽

Vol 75 (15) ◽

pp. 7206-7209 ◽

Cited By ~ 11

Author(s):

Vivien V. McDougal ◽

Linda A. Guarino

Keyword(s):

Conformational Change ◽

Atp Hydrolysis ◽

Protein Complexes ◽

Dna Helicase ◽

Atp Binding ◽

Dna Unwinding ◽

Single Stranded Dna ◽

Inchworm Mechanism

ABSTRACT P143 is a DNA helicase that tightly binds both double-stranded and single-stranded DNA. DNA-protein complexes rapidly dissociated in the presence of ATP and Mg2+. This finding suggests that ATP hydrolysis causes a conformational change in P143 which decreases affinity for DNA. This supports the model of an inchworm mechanism of DNA unwinding.

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ALKOXYALKYL ESTERS OF NUCLEOTIDE ANALOGS INHIBIT POLYOMAVIRUS DNA REPLICATION AND LARGE T ANTIGEN ACTIVITIES

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01641-20 ◽

2020 ◽

Author(s):

Nichodemus O. Onwubiko ◽

Suraya Diaz ◽

Marcela Krecmerova ◽

Heinz Peter Nasheuer

Keyword(s):

Dna Replication ◽

Tumor Antigens ◽

Antiviral Agent ◽

Dna Helicase ◽

T Antigen ◽

Nucleotide Analogs ◽

Large Tumor ◽

Nucleotide Analog ◽

Derivatives Of

Polyomavirus-related infections are ubiqutious in immunocompromised individuals and in some cases are intractable and fatal. Due to lack of approved drugs to treat polyomavirus infections, cidofovir, a phosphonate nucleotide analog approved to treat cytomegalovirus infections has been repurposed as anti-polyomavirus agent. Cidofovir has been modified in various ways to improve its efficacies as broad-spectrum antiviral agent. However, the actual mechanisms and targets of cidofovir and its modified derivatives as anti-polyomavirus agents are still under research. Here, polyomavirus large tumor antigens (Tag) activities were identified as the viral target of cidofovir derivatives. The alkoxyalkyl-ester derivatives of cidofovir efficiently inhibit polyomavirus DNA replication in cell-free human extracts and a viral in vitro replication system only utilizing purified proteins. We present evidence that DNA helicase, and DNA binding activities of polyomavirus Tags are diminished in the presence of low concentrations of alkoxyalkyl-ester derivatives of cidofovir suggesting that the inhibition of viral DNA replication is at least in part mediated by inhibiting ssDNA and dsDNA binding activities of Tags. These findings show that the alkoxyalkyl-ester derivatives of cidofovir are effective in vitro without undergoing further conversions and conclude that the inhibitory mechanisms of nucleotide analog-based drugs are more complex than previously believed.

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Synthetic DNA Replication Bubbles Bound and Unwound with Twofold Symmetry by a Simian Virus 40 T-Antigen Double Hexamer

Journal of Virology ◽

10.1128/jvi.72.11.8676-8681.1998 ◽

1998 ◽

Vol 72 (11) ◽

pp. 8676-8681 ◽

Cited By ~ 21

Author(s):

Natalia V. Smelkova ◽

James A. Borowiec

Keyword(s):

Dna Replication ◽

Simian Virus 40 ◽

Simian Virus ◽

Dna Helicase ◽

T Antigen ◽

Dna Unwinding ◽

Replication Forks ◽

Helicase Activity ◽

Synthetic Dna ◽

Stimulation Of

ABSTRACT Dimerization of simian virus 40 T-antigen hexamers (TAgH) into double hexamers (TAgDH) on model DNA replication forks has been found to greatly stimulate T-antigen DNA helicase activity. To explore the interaction of TAgDH with DNA during unwinding, we examined the binding of TAgDH to synthetic DNA replication bubbles. Tests of replication bubble substrates containing different single-stranded DNA (ssDNA) lengths indicated that efficient formation of a TAgDH requires ≥40 nucleotides (nt) of ssDNA. DNase I probing of a substrate containing a 60-nt ssDNA bubble complexed with a TAgDH revealed that T antigen bound the substrate with twofold symmetry. The strongest protection was observed over the 5′ junction on each strand, with 5 bp of duplex DNA and ∼17 nt of adjacent ssDNA protected from nuclease cleavage. Stimulation of the T-antigen DNA helicase activity by an increase in ATP concentration caused the protection to extend in the 5′ direction into the duplex region, while resulting in no significant changes to the 3′ edge of strongest protection. Our data indicate that each TAgH encircles one ssDNA strand, with a different strand bound at each junction. The process of DNA unwinding results in each TAgH interacting with a greater length of DNA than was initially bound, suggesting the generation of a more highly processive helicase complex.

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An emerging picture of FANCJ’s role in G4 resolution to facilitate DNA replication

NAR Cancer ◽

10.1093/narcan/zcab034 ◽

2021 ◽

Vol 3 (3) ◽

Author(s):

Robert M Brosh ◽

Yuliang Wu

Keyword(s):

Dna Replication ◽

Double Helix ◽

Dna Helicase ◽

Nucleic Acid Binding ◽

Dna Helicases ◽

Recent Advances ◽

G4 Dna ◽

Cellular Dna

Abstract A well-accepted hallmark of cancer is genomic instability, which drives tumorigenesis. Therefore, understanding the molecular and cellular defects that destabilize chromosomal integrity is paramount to cancer diagnosis, treatment and cure. DNA repair and the replication stress response are overarching paradigms for maintenance of genomic stability, but the devil is in the details. ATP-dependent helicases serve to unwind DNA so it is replicated, transcribed, recombined and repaired efficiently through coordination with other nucleic acid binding and metabolizing proteins. Alternatively folded DNA structures deviating from the conventional anti-parallel double helix pose serious challenges to normal genomic transactions. Accumulating evidence suggests that G-quadruplex (G4) DNA is problematic for replication. Although there are multiple human DNA helicases that can resolve G4 in vitro, it is debated which helicases are truly important to resolve such structures in vivo. Recent advances have begun to elucidate the principal helicase actors, particularly in cellular DNA replication. FANCJ, a DNA helicase implicated in cancer and the chromosomal instability disorder Fanconi Anemia, takes center stage in G4 resolution to allow smooth DNA replication. We will discuss FANCJ’s role with its protein partner RPA to remove G4 obstacles during DNA synthesis, highlighting very recent advances and implications for cancer therapy.

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Investigating the role of Rts1 in DNA replication initiation

Wellcome Open Research ◽

10.12688/wellcomeopenres.13884.1 ◽

2018 ◽

Vol 3 ◽

pp. 23 ◽

Cited By ~ 1

Author(s):

Ana B.A. Wallis ◽

Conrad A. Nieduszynski

Keyword(s):

Dna Replication ◽

Temperature Sensitivity ◽

Protein Phosphatase ◽

Dna Helicase ◽

Replication Initiation ◽

Replication Origins ◽

Origin Firing ◽

Replication Factor ◽

Dna Replication Initiation ◽

Genomic Screen

Background: Understanding DNA replication initiation is essential to understand the mis-regulation of replication seen in cancer and other human disorders. DNA replication initiates from DNA replication origins. In eukaryotes, replication is dependent on cell cycle kinases which function during S phase. Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) act to phosphorylate the DNA helicase (composed of mini chromosome maintenance proteins: Mcm2-7) and firing factors to activate replication origins. It has recently been found that Rif1 can oppose DDK phosphorylation. Rif1 can recruit protein phosphatase 1 (PP1) to dephosphorylate MCM and restricts origin firing. In this study, we investigate a potential role for another phosphatase, protein phosphatase 2A (PP2A), in regulating DNA replication initiation. The PP2A regulatory subunit Rts1 was previously identified in a large-scale genomic screen to have a genetic interaction with ORC2 (a DNA replication licensing factor). Deletion of RTS1 synthetically rescued the temperature-sensitive (ts-) phenotype of ORC2 mutants. Methods: We deleted RTS1 in multiple ts-replication factor Saccharomyces cerevisiae strains, including ORC2. Dilution series assays were carried out to compare qualitatively the growth of double mutant ∆rts1 ts-replication factor strains relative to the respective single mutant strains. Results: No synthetic rescue of temperature-sensitivity was observed. Instead we found an additive phenotype, indicating gene products function in separate biological processes. These findings are in agreement with a recent genomic screen which found that RTS1 deletion in several ts-replication factor strains led to increased temperature-sensitivity. Conclusions: We find no evidence that Rts1 is involved in the dephosphorylation of DNA replication initiation factors.

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Rad53 controls DNA unwinding after helicase-polymerase uncoupling at DNA replication forks

10.1101/811141 ◽

2019 ◽

Author(s):

Sujan Devbhandari ◽

Dirk Remus

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Point Mutations ◽

Dna Helicase ◽

Dna Unwinding ◽

Replication Forks ◽

Chromosomal Dna ◽

Replication Checkpoint ◽

Polymerase Domain ◽

Tightly Coupled

ABSTRACTThe coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Using the reconstituted budding yeast DNA replication system, we demonstrate that Pol ε variants harboring catalytic point mutations in the Pol2 polymerase domain, contrary to Pol2 polymerase domain deletions, inhibit DNA synthesis at replication forks by displacing Pol δ from PCNA/primer-template junctions, causing excessive DNA unwinding by the replicative DNA helicase, CMG, uncoupled from DNA synthesis. Mutations that suppress the inhibition of Pol δ by Pol ε restore viability in Pol2 polymerase point mutant cells. We also observe uninterrupted DNA unwinding at replication forks upon dNTP depletion or chemical inhibition of DNA polymerases, demonstrating that leading strand synthesis is not tightly coupled to DNA unwinding by CMG. Importantly, the Rad53 kinase controls excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork-stabilization by the replication checkpoint.

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Mechanistic insights into Lhr helicase function in DNA repair

Biochemical Journal ◽

10.1042/bcj20200379 ◽

2020 ◽

Vol 477 (16) ◽

pp. 2935-2947

Author(s):

Ryan J. Buckley ◽

Kevin Kramm ◽

Christopher D. O. Cooper ◽

Dina Grohmann ◽

Edward L. Bolt

Keyword(s):

Dna Repair ◽

Dna Replication ◽

Single Molecule ◽

Dna Helicase ◽

Holliday Junctions ◽

Single Molecule Fret ◽

Repair Enzymes ◽

Dna Strands

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared with DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the ‘parental’ DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication.

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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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