Fork sensing and strand switching control antagonistic activities of RecQ helicases
TL;DR: This work investigates the DNA unwinding of RecQ helicases from Arabidopsis thaliana, AtRECQ2 and AtRECZ3 at the single-molecule level using magnetic tweezers and provides a simple explanation for how different biological activities can be achieved by rather similar members of the RecQ family.
Abstract: RecQ helicases have essential roles in maintaining genome stability during replication and in controlling double-strand break repair by homologous recombination. Little is known about how the different RecQ helicases found in higher eukaryotes achieve their specialized and partially opposing functions. Here, we investigate the DNA unwinding of RecQ helicases from Arabidopsis thaliana, AtRECQ2 and AtRECQ3 at the single-molecule level using magnetic tweezers. Although AtRECQ2 predominantly unwinds forked DNA substrates in a highly repetitive fashion, AtRECQ3 prefers to rewind, that is, to close preopened DNA forks. For both enzymes, this process is controlled by frequent strand switches and active sensing of the unwinding fork. The relative extent of the strand switches towards unwinding or towards rewinding determines the predominant direction of the enzyme. Our results provide a simple explanation for how different biological activities can be achieved by rather similar members of the RecQ family.
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TL;DR: This work shows that, akin to the Mus81-Mms4, Yen1, and MutLγ-Exo1 nucleases, Sgs1 helicase function is under cell-cycle control through the actions of CDK and Cdc5 kinases, and suggests a concerted mechanism driving orderly formation of noncrossover and crossover recombinants in meiotic and mitotic cells.
23 citations
Cites background from "Fork sensing and strand switching c..."
...This behavior has been previously observed for Sgs1, as well as other eukaryotic RecQ helicases (Kasaciunaite et al., 2019; Klaue et al., 2013)....
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TL;DR: It is shown that WRN alone is a weak helicase which repetitively unwind just a few tens of base pairs, but that binding of multiple RPAs to the enzyme converts WRN into a superhelicase that unidirectionally unwinds double-stranded DNA more than 1 kb.
Abstract: RPA is known to stimulate the helicase activity of Werner syndrome protein (WRN), but the exact stimulation mechanism is not understood. We use single-molecule FRET and magnetic tweezers to investigate the helicase activity of WRN and its stimulation by RPA. We show that WRN alone is a weak helicase which repetitively unwind just a few tens of base pairs, but that binding of multiple RPAs to the enzyme converts WRN into a superhelicase that unidirectionally unwinds double-stranded DNA more than 1 kb. Our study provides a good case in which the activity and biological functions of the enzyme may be fundamentally altered by the binding of cofactors.
23 citations
Cites result from "Fork sensing and strand switching c..."
...However, according to this work and the previous magnetic tweezers studies on BLM and AtRecQ2 (32,34), the critical length it unwinds at a time was not narrowly distributed as proposed and it was considerably affected by factors such as base composition and applied force....
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...This pattern is similar to those reported for BLM (a member of human RecQ) and Arabidopsis thaliana RecQ (AtRecQ2) (32,34)....
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TL;DR: The dynamics of DNA unwinding by Sgs1 at the single‐molecule level are resolved and it is found that Dna2 modulates the velocity of Sgs2, indicating that during end resection both proteins form a functional complex and couple their activities.
Abstract: DNA double-strand break repair by homologous recombination employs long-range resection of the 5' 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.
22 citations
Cites background or result from "Fork sensing and strand switching c..."
...The observed behavior was similar to that seen for BLM (Wang et al, 2015), WRN (Lee et al, 2018), and the likely BLM homolog from Arabidopsis thaliana, AtRecQ2 (Klaue et al, 2013)....
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...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, 2013)....
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...As a result, the enzyme is bound in a more loosely state since it lacks the DNA junction in its wake (Klaue et al, 2013)....
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...Due to the functional similarities between Sgs1, BLM, and AtRecQ2 (Oh et al, 2007; De Muyt et al, 2012; Klaue et al, 2013), we suggest that Sgs1 also undergoes cycles of strand switches during repetitive DNA unwinding–rezipping events, suggesting that this is a conserved characteristic of RecQ helicases....
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...For AtRecQ2, the transition between unwinding and rezipping most likely involves strand switching (Klaue et al, 2013)....
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TL;DR: It is concluded that, as in fungi, AtHRQ1 has a conserved function in DNA excision repair and not only shares pathways with the Fanconi anemia repair factors, but in contrast to fungi also seems to act in a common pathway with postreplicative DNA repair.
Abstract: RecQ helicases are important caretakers of genome stability and occur in varying copy numbers in different eukaryotes. Subsets of RecQ paralogs are involved in DNA crosslink (CL) repair. The orthologs of AtRECQ2, AtRECQ3 and AtHRQ1, HsWRN, DmRECQ5 and ScHRQ1 participate in CL repair in their respective organisms, and we aimed to define the function of these helicases for plants. We obtained Arabidopsis mutants of the three RecQ helicases and determined their sensitivity against CL agents in single- and double-mutant analyses. Only Athrq1, but not Atrecq2 and Atrecq3, mutants proved to be sensitive to intra- and interstrand crosslinking agents. AtHRQ1 is specifically involved in the repair of replicative damage induced by CL agents. It shares pathways with the Fanconi anemia-related endonuclease FAN1 but not with the endonuclease MUS81. Most surprisingly, AtHRQ1 is epistatic to the ATPase RAD5A for intra- as well as interstrand CL repair. We conclude that, as in fungi, AtHRQ1 has a conserved function in DNA excision repair. Additionally, HRQ1 not only shares pathways with the Fanconi anemia repair factors, but in contrast to fungi also seems to act in a common pathway with postreplicative DNA repair.
20 citations
Cites background from "Fork sensing and strand switching c..."
...Recent single-molecule analyses underlined their different functionalization by uncovering a highly repetitive DNA unwinding activity for RECQ2, while RECQ3 preferably rewinds DNA forks (Klaue et al., 2013)....
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TL;DR: The marked difference in effect of the translocating strand cPu on rate of DNA unwinding between DDX11 and FANCJ helicase suggests the two Fe-S cluster helicases unwind damaged DNA by distinct mechanisms.
Abstract: 8,5′ cyclopurine deoxynucleosides (cPu) are locally distorting DNA base lesions corrected by nucleotide excision repair (NER) and proposed to play a role in neurodegeneration prevalent in genetically defined Xeroderma pigmentosum (XP) patients. In the current study, purified recombinant helicases from different classifications based on sequence homology were examined for their ability to unwind partial duplex DNA substrates harboring a single site-specific cPu adduct. Superfamily (SF) 2 RecQ helicases (RECQ1, BLM, WRN, RecQ) were inhibited by cPu in the helicase translocating strand, whereas helicases from SF1 (UvrD) and SF4 (DnaB) tolerated cPu in either strand. SF2 Fe-S helicases (FANCJ, DDX11 (ChlR1), DinG, XPD) displayed marked differences in their ability to unwind the cPu DNA substrates. Archaeal Thermoplasma acidophilum XPD (taXPD), homologue to the human XPD helicase involved in NER DNA damage verification, was impeded by cPu in the non-translocating strand, while FANCJ was uniquely inhibited by the cPu in the translocating strand. Sequestration experiments demonstrated that FANCJ became trapped by the translocating strand cPu whereas RECQ1 was not, suggesting the two SF2 helicases interact with the cPu lesion by distinct mechanisms despite strand-specific inhibition for both. Using a protein trap to simulate single-turnover conditions, the rate of FANCJ or RECQ1 helicase activity was reduced 10-fold and 4.5-fold, respectively, by cPu in the translocating strand. In contrast, single-turnover rates of DNA unwinding by DDX11 and UvrD helicases were only modestly affected by the cPu lesion in the translocating strand. The marked difference in effect of the translocating strand cPu on rate of DNA unwinding between DDX11 and FANCJ helicase suggests the two Fe-S cluster helicases unwind damaged DNA by distinct mechanisms. The apparent complexity of helicase encounters with an unusual form of oxidative damage is likely to have important consequences in the cellular response to DNA damage and DNA repair.
20 citations
Cites background from "Fork sensing and strand switching c..."
...The selective deterrence of DNA unwinding by the RecQ helicases when the cPu lesion resided in the translocating strand is interesting in light of recent experimental findings that BLM [58] and Arabidopsis RecQ homologs [59] have the ability to switch strands upon encountering undamaged double-stranded DNA and effectively translocate on the opposite strand away from the duplex....
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References
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TL;DR: This review sets out to define a nomenclature for helicase and translocase enzymes based on current knowledge of sequence, structure, and mechanism, and delineate six superfamilies of enzymes, with examples of crystal structures where available.
Abstract: Helicases and translocases are a ubiquitous, highly diverse group of proteins that perform an extraordinary variety of functions in cells. Consequently, this review sets out to define a nomenclature for these enzymes based on current knowledge of sequence, structure, and mechanism. Using previous definitions of helicase families as a basis, we delineate six superfamilies of enzymes, with examples of crystal structures where available, and discuss these structures in the context of biochemical data to outline our present understanding of helicase and translocase activity. As a result, each superfamily is subdivided, where appropriate, on the basis of mechanistic understanding, which we hope will provide a framework for classification of new superfamily members as they are discovered and characterized.
1,145 citations
TL;DR: Two different structures of PcrA DNA helicase complexed with the same single strand tailed DNA duplex are determined, providing snapshots of different steps on the catalytic pathway, providing evidence against an "active rolling" model for helicase action but are instead consistent with an "inchworm" mechanism.
759 citations
TL;DR: The structure of the HCV NS3 RNA helicase domain complexed with a single-stranded DNA oligonucleotide has been solved to 2.2 A resolution and is a member of a superfamily of helicases, termed superfamily II.
630 citations
TL;DR: This Review discusses how these proteins might suppress genomic rearrangements, and therefore function as 'caretaker' tumour suppressors.
Abstract: Around 1% of the open reading frames in the human genome encode predicted DNA and RNA helicases. One highly conserved group of DNA helicases is the RecQ family. Genetic defects in three of the five human RecQ helicases, BLM, WRN and RECQ4, give rise to defined syndromes associated with cancer predisposition, some features of premature ageing and chromosomal instability. In recent years, there has been a tremendous advance in our understanding of the cellular functions of individual RecQ helicases. In this Review, we discuss how these proteins might suppress genomic rearrangements, and therefore function as 'caretaker' tumour suppressors.
432 citations
TL;DR: A series of crystal structures of the UvrD helicase complexed with DNA and ATP hydrolysis intermediates reveal that ATP binding alone leads to unwinding of 1 base pair by directional rotation and translation of the DNA duplex, and ADP and Pi release leads to translocation of the developing single strand.
338 citations