Dynamics of the Eukaryotic Replicative Helicase at Lagging-Strand Protein Barriers Support the Steric Exclusion Model

Covalent DNA protein crosslinks (DPCs) are common lesions that block replication. We examine here the consequence of DPCs on mutagenesis involving replicational template-switch reactions in Escherichia coli. 5-azacytidine (5azaC) is a potent mutagen for template-switching, dependent on DNA cytosine methylase (Dcm), implicating the trapped Dcm-DNA covalent complex as the initiator for mutagenesis. The leading strand of replication is more mutable than the lagging strand, explained by blocks to the replicative helicase and/or fork regression. We find that template-switch mutagenesis induced by 5-azaC does not require DSB repair via RecABCD. The ability to induce the SOS response is anti-mutagenic by an unknown mechanism. Mutants in recB, but not recA, exhibit high constitutive rates of template-switching and we suggest that RecBCD-mediated DNA degradation prevents template-switching associated with fork regression. A mutation in the DnaB fork helicase also promotes high levels of template-switching. We also find that other DPC-inducers, formaldehyde (a non-specific crosslinker) and ciprofloxacin (a topoisomerase II poison) are also strong mutagens for template-switching. Induction of mutations and genetic rearrangements that occur by template-switching may constitute a previously unrecognized component of the genotoxicity and genetic instability promoted by DPCs.

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Multistep mechanism of DNA replication-coupled G-quadruplex resolution

10.1101/2020.11.11.378067 ◽

2020 ◽

Author(s):

Koichi Sato ◽

Nerea Martin-Pintado ◽

Harm Post ◽

Maarten Altelaar ◽

Puck Knipscheer

Keyword(s):

Dna Replication ◽

Genome Stability ◽

Genome Instability ◽

Genome Maintenance ◽

Replicative Helicase ◽

Dna Structures ◽

G Quadruplex ◽

Egg Extracts ◽

Leading Strand ◽

Lagging Strand

SummaryG-quadruplex (or G4) structures are non-canonical DNA structures that form in guanine-rich sequences and threaten genome stability when not properly resolved. G4 unwinding occurs during S phase via an unknown mechanism. Using Xenopus egg extracts, we define a three-step G4 unwinding mechanism that is coupled to DNA replication. First, the replicative helicase (CMG) stalls at a leading strand G4 structure. Second, the DHX36 helicase mediates the bypass of the CMG past the intact G4 structure, which allows approach of the leading strand to the G4. Third, G4 structure unwinding by the FANCJ helicase enables the DNA polymerase to synthesize past the G4 motif. A G4 on the lagging strand template does not stall CMG, but still requires DNA replication for unwinding. DHX36 and FANCJ have partially redundant roles, conferring robustness to this pathway. Our data reveal a novel genome maintenance pathway that promotes faithful G4 replication thereby avoiding genome instability.

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Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase

eLife ◽

10.7554/elife.23449 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 30

Author(s):

Lance Langston ◽

Mike O’Donnell

Keyword(s):

Exclusion Process ◽

The Other ◽

Dna Unwinding ◽

Duplex Dna ◽

Central Channel ◽

Replicative Helicase ◽

Steric Exclusion ◽

Dna Strand

Replicative helicases are ring-shaped hexamers that encircle DNA for duplex unwinding. The currently accepted view of hexameric helicase function is by steric exclusion, where the helicase encircles one DNA strand and excludes the other, acting as a wedge with an external DNA unwinding point during translocation. Accordingly, strand-specific blocks only affect these helicases when placed on the tracking strand, not the excluded strand. We examined the effect of blocks on the eukaryotic CMG and, contrary to expectations, blocks on either strand inhibit CMG unwinding. A recent cryoEM structure of yeast CMG shows that duplex DNA enters the helicase and unwinding occurs in the central channel. The results of this report inform important aspects of the structure, and we propose that CMG functions by a modified steric exclusion process in which both strands enter the helicase and the duplex unwinding point is internal, followed by exclusion of the non-tracking strand.

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Involvement of a replicative DNA helicase of bacteriophage T4 in DNA recombination.

Genetics ◽

10.1093/genetics/138.2.247 ◽

1994 ◽

Vol 138 (2) ◽

pp. 247-252 ◽

Cited By ~ 3

Author(s):

T Yonesaki

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Genetic Recombination ◽

Bacteriophage T4 ◽

Dna Recombination ◽

Dna Helicase ◽

Replicative Helicase ◽

Helicase Gene ◽

A Subunit ◽

Lagging Strand

Abstract Bacteriophage T4 gene 41 encodes a replicative DNA helicase that is a subunit of the primosome which is essential for lagging-strand DNA synthesis. A mutation, rrh, was generated and selected in the helicase gene on the basis of limited DNA replication that ceases early. The survival of ultraviolet-irradiated phage and the frequency of genetic recombination are reduced by rrh. In addition, rrh diminishes the production of concatemeric DNA. These results strongly suggest that the gene 41 replicative helicase participates in DNA recombination.

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Mcm10 functions to isomerize CMG-DNA for replisome bypass of DNA blocks

10.1101/146837 ◽

2017 ◽

Cited By ~ 1

Author(s):

Lance D. Langston ◽

Ryan Mayle ◽

Grant D. Schauer ◽

Olga Yurieva ◽

Daniel Zhang ◽

...

Keyword(s):

Cell Types ◽

The Other ◽

Replication Protein ◽

Duplex Dna ◽

Replication Forks ◽

Central Channel ◽

Steric Exclusion ◽

Dna Complex ◽

Lagging Strand

AbstractReplicative helicases of all cell types are rings that unwind DNA by steric exclusion in which the helicase ring only encircles the tracking strand, excluding the other strand outside the ring. Steric exclusion mediated unwinding enables helicase rings to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG encircles duplex DNA at a forked junction and is stopped by a block on the non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential replication protein unique to eukaryotes, binds CMG and enables the replisome to bypass blocks on the non-tracking strand, implying that Mcm10 isomerizes the CMG-DNA complex to position only one strand through the central channel. A similar CMG-DNA isomerization is needed at the origin for head-to-head CMGs to bypass one another during formation of bidirectional replication forks.

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Mcm10 promotes rapid isomerization of CMG-DNA for replisome bypass of lagging strand DNA blocks

eLife ◽

10.7554/elife.29118 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 41

Author(s):

Lance D Langston ◽

Ryan Mayle ◽

Grant D Schauer ◽

Olga Yurieva ◽

Daniel Zhang ◽

...

Keyword(s):

Cell Types ◽

The Other ◽

Replication Protein ◽

Helicase Activity ◽

Central Channel ◽

Steric Exclusion ◽

Dna Strand ◽

Dna Complex ◽

Lagging Strand

Replicative helicases in all cell types are hexameric rings that unwind DNA by steric exclusion in which the helicase encircles the tracking strand only and excludes the other strand from the ring. This mode of translocation allows helicases to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG helicase partially encircles duplex DNA at a forked junction and is stopped by a block on the non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential replication protein unique to eukaryotes, binds CMG and greatly stimulates its helicase activity in vitro. Most significantly, Mcm10 enables CMG and the replisome to bypass blocks on the non-tracking DNA strand. We demonstrate that bypass occurs without displacement of the blocks and therefore Mcm10 must isomerize the CMG-DNA complex to achieve the bypass function.

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