Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination

Harami, Gábor M.; Seol, Yeonee; In, Junghoon; Ferencziová, Veronika; Martina, Máté; Gyimesi, Máté; Sarlós, Kata; Kovács, Zoltán; Nagy, Nikolett; Sun, Yuze; Vellai, Tibor; Neuman, Keir C.; Kovács, Mihály

doi:10.1073/pnas.1615439114

Cited by 39 publications

(96 citation statements)

References 61 publications

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“…These RPA variants failed to stimulate helicase initiation (Figure 3E). More strikingly, all three variants-but not wt RPA-induced the intrinsic strand-switching activity previously reported for BLM and other RecQ helicases (Figure 3D,F and S3D) (Chen and Brill, 2014;Harami et al, 2017;Klaue et al, 2013;Wang et al, 2015;Yodh et al, 2009). Strand-switching refers to BLM alternating between translocation on either the Watson or Crick ssDNA strands (Wang et al, 2015;Yodh et al, 2009).…”

Section: Rpa70n Regulates Blm Strand-switchingsupporting

confidence: 54%

RPA phosphorylation regulates DNA resection

Soniat

Paull

et al. 2019

Preprint

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Genetic recombination in all kingdoms of life initiates when helicases and nucleases process (resect) the free DNA ends to expose single-stranded (ss) DNA overhangs. Resection termination in bacteria is programmed by a DNA sequence but the mechanisms limiting resection in eukaryotes have remained elusive. Using single-molecule imaging of reconstituted human DNA repair factors, we identify a general mechanism that limits DNA resection. BLM helicase together with EXO1 and DNA2 nucleases catalyze kilobase-length DNA resection on nucleosome-coated DNA. The resulting ssDNA is rapidly bound by RPA, which is in turn phosphorylated as part of the DNA damage response (DDR). Remarkably, phosphorylated RPA (pRPA) inhibits DNA resection via regulation of BLM helicase. pRPA suppresses BLM initiation at DNA ends and promotes the intrinsic helicase strand-switching activity. These findings establish that pRPA is a critical regulator of DNA repair enzymes and provides a feedback loop between the DDR and DNA resection termination.

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Section: Rpa70n Regulates Blm Strand-switchingsupporting

confidence: 54%

RPA phosphorylation regulates DNA resection

Soniat

Paull

et al. 2019

Preprint

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“…DNA unwinding by Sgs1 was found to be highly dynamic, involving many repetitive unwinding events separated by either rapid DNA rezipping or slower DNA rewinding. Sgs1 shares this highly dynamic activity pattern with other RecQ family helicases from prokaryotes (Harami et al , ; Bagchi et al , ) and eukaryotes (Klaue et al , ; Wang et al , ; Lee et al , ). It is thought that the switching between unwinding and rewinding involves repeated strand switching events to allow direction reversals of the helicase (Klaue et al , ).…”

Section: Discussionmentioning

confidence: 92%

Competing interaction partners modulate the activity of Sgs1 helicase during DNA end resection

et al. 2019

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DNA double-strand break repair by homologous recombination employs long-range resection of the 5 0 DNA ends at the break points. In Saccharomyces cerevisiae, this process can be performed by the RecQ helicase Sgs1 and the helicase-nuclease Dna2. Though functional interplay between them has been shown, it remains unclear whether and how these proteins cooperate on the molecular level. Here, we resolved the dynamics of DNA unwinding by Sgs1 at the single-molecule level and investigated Sgs1 regulation by Dna2, the single-stranded DNA-binding protein RPA, and the Top3-Rmi1 complex. We found that Dna2 modulates the velocity of Sgs1, indicating that during end resection both proteins form a functional complex and couple their activities. Sgs1 drives DNA unwinding and feeds single-stranded DNA to Dna2 for degradation. RPA was found to regulate the processivity and the affinity of Sgs1 to the DNA fork, while Top3-Rmi1 modulated the velocity of Sgs1. We hypothesize that the differential regulation of Sgs1 activity by its protein partners is important to support diverse cellular functions of Sgs1 during the maintenance of genome stability.

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“…The Sgs1 C-terminus contains a region predicted to be highly unstructured. This region follows immediately after the conserved HRDC (Helicase-and-RNaseD-like-C-terminal) domain, which is important for substrate binding and regulation of helicase activity (LIU et al 1999;YANKIWSKI et al 2001;HICKSON 2003;WU et al 2005;CHU AND HICKSON 2009;VINDIGNI AND HICKSON 2009;HARAMI et al 2017). The HA epitope has a net negative charge, and could conceivably interfere with Sgs1-DNA interaction, especially in the HRDC domain.…”

Section: Discussionmentioning

confidence: 99%

C-terminal HA tags compromise function and exacerbate phenotypes ofSaccharomyces cerevisiaeBloom’s helicase homolog Sgs1 SUMOylation-associated mutants

Lichten

Cohen

2020

Preprint

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The Sgs1 helicase and Top3-Rmi1 decatenase form a complex that affects homologous recombination outcomes during the mitotic cell cycle and during meiosis. Previous studies have reported that Sgs1-Top3-Rmi1 function is regulated by SUMOylation that is catalyzed by the Smc5-Smc6-Mms21 complex. These studies used strains in which SGS1 was C-terminally tagged with three or six copies of a human influenza hemagglutinin-derived epitope tag (3HA and 6HA). They identified SGS1 mutants that affect its SUMOylation, which we will refer to as SGS1 SUMO-site mutants. In previous work, these mutants showed phenotypes consistent with substantial loss of Sgs1-Top3-Rmi1 function during the mitotic cell cycle. We find that the reported phenotypes are largely due to the presence of the HA epitope tags. Untagged SGS1 SUMO-site mutants show either wild-type or weak hypomorphic phenotypes, depending on the assay. These phenotypes are exacerbated by both 6HA and 3HA epitope tags in two different S. cerevisiae strain backgrounds. Importantly, a C-terminal 6HA tag confers strong hypomorphic or null phenotypes on an otherwise wild-type Sgs1 protein. Taken together, these results suggest that the HA epitope tags used in previous studies seriously compromise Sgs1 function. Furthermore, they raise the possibilities either that sufficient SUMOylation of the Sgs1-Top3-Rmi1 complex might still occur in the SUMO-site mutants isolated, or that Smc5-Smc6-Mms21mediated SUMOylation plays a minor role in the regulation of Sgs1-Top3-Rmi1 during recombination.

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Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination

Cited by 39 publications

References 61 publications

RPA phosphorylation regulates DNA resection

RPA phosphorylation regulates DNA resection

Competing interaction partners modulate the activity of Sgs1 helicase during DNA end resection

C-terminal HA tags compromise function and exacerbate phenotypes ofSaccharomyces cerevisiaeBloom’s helicase homolog Sgs1 SUMOylation-associated mutants

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