A Hexameric Helicase Encircles One DNA Strand and Excludes the Other during DNA Unwinding†

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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Interaction of HelQ helicase with RPA modulates RPA-DNA binding and stimulates HelQ to unwind DNA through a protein roadblock

10.1101/511758 ◽

2019 ◽

Author(s):

Sarah J. Northall ◽

Tabitha Jenkins ◽

Denis Ptchelkine ◽

Vincenzo Taresco ◽

Christopher D. O. Cooper ◽

...

Keyword(s):

Dna Binding ◽

Dna Unwinding ◽

Abasic Site ◽

Domain Interaction ◽

Dna Helicases ◽

Intrinsically Disordered ◽

Catalytically Active ◽

Dna Strands ◽

Dna Strand ◽

Protein Barrier

ABSTRACTCells reactivate compromised DNA replication forks using enzymes that include DNA helicases for separating DNA strands and remodelling protein-DNA complexes. HelQ helicase promotes replication-coupled DNA repair in mammals in a network of interactions with other proteins. We report newly identified HelQ helicase activities, when acting alone and when interacting with RPA. HelQ helicase was strongly inhibited by a DNA-protein barrier (BamHIE111A), and by an abasic site in the translocating DNA strand. Interaction of HelQ with RPA activated DNA unwinding through the protein barrier, but not through the abasic site. Activation was lost when RPA was replaced with bacterial SSB or DNA binding-defective RPA, RPAARO1. We observed stable HelQ-RPA-DNA ternary complex formation, and present evidence that an intrinsically disordered N-terminal region of HelQ (N-HelQ) interacts with RPA, destabilising RPA-DNA binding. Additionally, SEC-MALS showed that HelQ multimers are converted into catalytically active dimers when ATP-Mg2+ is bound. HelQ and RPA are proposed to jointly promote replication fork recovery by helicase-catalysed displacement of DNA-bound proteins, after HelQ gains access to ssDNA through its N-terminal domain interaction with RPA.

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

10.1101/796003 ◽

2019 ◽

Author(s):

Hazal B. Kose ◽

Sherry Xie ◽

George Cameron ◽

Melania S. Strycharska ◽

Hasan Yardimci

Keyword(s):

Single Molecule ◽

Replication Fork ◽

Double Helix ◽

Dna Unwinding ◽

Helicase Activity ◽

Replicative Helicase ◽

Double Stranded Dna ◽

Order Of Magnitude ◽

Dna Strand ◽

Dna Double Helix

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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Genetic evidence for preferential strand transfer during meiotic recombination in yeast.

Genetics ◽

10.1093/genetics/125.4.753 ◽

1990 ◽

Vol 125 (4) ◽

pp. 753-761 ◽

Cited By ~ 1

Author(s):

D K Nag ◽

T D Petes

Keyword(s):

Saccharomyces Cerevisiae ◽

Meiotic Recombination ◽

Exchange Process ◽

Genetic Evidence ◽

The Other ◽

Strand Transfer ◽

Yeast Saccharomyces Cerevisiae ◽

Dna Strand

Abstract During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplexes are formed as an intermediate in the exchange process. In the formation of an asymmetric heteroduplex, one chromosome acts as a donor of a single DNA strand and the other acts as a recipient. We present genetic evidence that the nontranscribed strand is donated more frequently than the transcribed strand in spores that have an unrepaired mismatch at the HIS4 locus.

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Copy choice mechanism of immunoglobulin heavy-chain switch recombination.

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.397 ◽

1990 ◽

Vol 10 (1) ◽

pp. 397-400 ◽

Cited By ~ 34

Author(s):

W Dunnick ◽

J Stavnezer

Keyword(s):

Heavy Chain ◽

Recombination Event ◽

Immunoglobulin Heavy Chain ◽

The Other ◽

Constant Region ◽

Switch Regions ◽

Dna Strand

The immunoglobulin heavy-chain switch is mediated by a recombination event between DNA switch regions associated with donor and recipient constant-region genes. We have determined that the mutations which can be found in some switch regions after recombination appear to arise on only one strand of DNA. This result suggests that switch recombination involves error-prone synthesis of one DNA strand and ligation of the other strand from preexisting DNA.

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