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DNA Helicase-SSB Interactions Critical to the Regression and Restart of Stalled DNA Replication Forks in Escherichia Coli

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G C A T T A C G G C A T genes Review DNA Helicase-SSB Interactions Critical to the Regression and Restart of Stalled DNA Replication Forks in Escherichia coli Piero R. Bianco Center for Single Molecule Biophysics, University at Buffalo, SUNY, Buffalo, NY 14221, USA; pbianco@buffalo.edu; Tel.: +(716)-829-2599 Received: 31 March 2020; Accepted: 23 April 2020; Published: 26 April 2020 Abstract: In Escherichia coli, DNA replication forks stall on average once per cell cycle. When this occurs, replisome components disengage from the DNA, exposing an intact, or nearly intact fork. Consequently, the fork structure must be regressed away from the initial impediment so that repair can occur. Regression is catalyzed by the powerful, monomeric DNA helicase, RecG. During this reaction, the enzyme couples unwinding of fork arms to rewinding of duplex DNA resulting in the formation of a Holliday junction. RecG works against large opposing forces enabling it to clear the fork of bound proteins. Following subsequent processing of the extruded junction, the PriA helicase mediates reloading of the replicative helicase DnaB leading to the resumption of DNA replication. The single-strand binding protein (SSB) plays a key role in mediating PriA and RecG functions at forks. It binds to each enzyme via linker/OB-fold interactions and controls helicase-fork loading sites in a substrate-dependent manner that involves helicase remodeling. Finally, it is displaced by RecG during fork regression. The intimate and dynamic SSB-helicase interactions play key roles in ensuring fork regression and DNA replication restart. Keywords: RecG; single-strand binding protein (SSB); Stalled DNA replication fork; DNA repair; DNA replication; helicase; atomic force microscopy; OB-fold; SH3 domain; PXXP motif 1. Introduction The precise duplication of the genome relies on the DNA replication, DNA repair and genetic recombination machinery working closely together [1–5]. This complex interplay is required as the advancing replisomes frequently encounter roadblocks that have the potential to stall or collapse replication forks [6,7]. In Escherichia coli, forks stall on average at least once per cell cycle and require restart. The types of obstructions to fork progression include proteins bound to the parental duplex, non-coding lesions in the template DNA, unusual secondary structures that arise in the DNA and either single- or double-strand breaks [3,8–12]. Each of these roadblocks to DNA replication can use different subsets of repair enzymes and this is highlighted by the varied recombination and repair gene requirements for fork rescue [9,13–17]. For example, when the replisome encounters a nick in the leading strand, the structure of the fork will collapse and require the processing of the nascent double-strand break by RecBCD followed by strand invasion catalyzed by RecA. This results in the formation of a displacement loop that is then used to reload the replicative helicase DnaB leading to the restart of DNA replication [15]. In contrast, when replisomes encounter an impediment such as a dsDNA crosslink or bound proteins such as RNA polymerase or mutant methyltransferases, a stalled DNA replication fork is formed [18]. A stalled fork can be directly restarted or reversed (regressed; Figure1 and [ 7,9,13,19–23]). Consequently, regression occurs in a direction opposite to that of replication, with the fork actively Genes 2020, 11, 471; doi:10.3390/genes11050471 www.mdpi.com/journal/genes Genes 2020, 11, 471 2 of 18 Genes 2020, 11, x FOR PEER REVIEW 2 of 18 moved away from the site of damage to a region where the nascent, replicated genome is unharmed. This actively moved away from the site of damage to a region where the nascent, replicated genome is enables the repair machinery to access the obstruction and facilitate repair. This process is analogous unharmed. This enables the repair machinery to access the obstruction and facilitate repair. This to the clearing of train tracks following a collision and possible derailment. process is analogous to the clearing of train tracks following a collision and possible derailment. Figure 1.1. ForkFork regression. regression. (A ).(A Fork). Fork regression regression occurs occurs in a direction in a direction opposite opposite to that of to DNA that replication. of DNA Anreplication. impeded An fork impeded is shown fork with is shown the nascent with the leading nascent and leading lagging and strands lagging coloredstrands bluecolored and blue orange, and orange,respectively. respectively. Fork regression Fork regr involvesession unwindinginvolves unwinding of these nascentof these duplex nascent regions, duplex concomitant regions , withconcomitant the extrusion with the of twoextrusion duplex ofregions two duplex colored regi inons green. colored The in resulting green. The DNA resulting structure DNA is knownstructure as isthe known “chicken as foot” the “chicken and is structurally foot” and similar is structurally to a Holliday similar junction. to a Fork Holliday reversal junction. (indicated Fork by thereversal balck (indicatedarrow) results by the inleftward balck arrow) movement results of in the leftward fork and movement an occur onlyof the once fork regression and an occur and subsequent only once regressionimpediment and repair subsequent have taken impedime palce. (Bnt). Forkrepair regression have taken requires palce. that (B). DNA Fork unwindingregression berequires coupled that to DNAduplex unwinding rewinding. be (C coupled). The RecG to duplex DNA helicaserewinding. is shown (C). The in the RecG process DNA of helicase fork regression is shown where in the it processcouples of DNA fork unwinding regression towhere duplex it couples rewinding DNA and unwinding displacement to duplex of the single-strandrewinding and binding displacement protein (SSB)of the single-strand protein. RecG binding and SSB protei aren represented(SSB) protein. as RecG Connolly and SSB surfaces. are represented The wedge as Connolly domain surfaces. of RecG Theis colored wedge purple domain and of the RecG helicase is colored domains purple in cyan. and the SSB helicase monomers domains are colored in cyan. orange SSB monomers and green. are colored orange and green. Which replisome proteins are disengaged from the DNA [24,25]? At a minimum, this must be DnaB[24,25] because Which of the replisome requirement proteins for theare restartdisengaged DNA from helicase, the DNA? PriA [ 26At– 28a minimum,]. However, this due must to thebe DnaBability because of polymerases of the requirement to rapidly turn for over,the restart it is conceivable DNA helicase, that thesePriA components,[26–28]. However, as well due as others,to the abilitymight disengageof polymerases as well to [ 29rapidly,30]. However, turn over, protein it is conceivable disengagement that these may notcomponents, always occur as well as recent as others, work mightfrom the disengage Marians as group well has [29,30]. shown However, [31]. In thisprotein study, disengagement they demonstrated may not that always the replisome occur as remains recent workstably from associated the Marians with the group fork afterhas shown a collision [31]. with In th ais leading-strand study, they demonstrated template lesion. that Leading-strand the replisome remainsDNA synthesis stably wasassociated then reinitiated with the downstreamfork after a ofcollision the damage with ina aleading-strand reaction that istemplate independent lesion. of Leading-strandany of the known DNA replication-restart synthesis was proteins.then reinitiated downstream of the damage in a reaction that is independentIf one or of more any componentsof the known of replication-restart the replisome disengage proteins. from the DNA, then replication cannot progressIf one and or themore fork components becomes stalled. of the replisome Regression di ofsengage the stalled from DNA the DNA, replication then replication fork likely cannot occurs (Figureprogress1A). and This the can fork be becomes driven by stalled. accumulated Regression positive of superhelicalthe stalled DNA tension replication as shown fork by the likely Cozzarelli occurs (Figuregroup [32 1A).,33]. This It could can alsobe driven be catalyzed by accumulated by several proteins positive including superhelical the recombinase tension as RecA,shown the by DNA the Cozzarellihelicase RecG group and [32,33]. the resolvase It could RuvAB also [be34 –catalyzed39]. However, by several the evidence proteins overwhelmingly including the demonstratesrecombinase RecA,that RecG the catalyzes DNA helicase fork regression RecG asand explained the resolvase below. RuvAB [34–39]. However, the evidence overwhelminglyRecA is unlikely demonstrates to be involved that RecG in fork cata regressionlyzes fork regression where either as theexplained leading below. or lagging strands containRecA exposed is unlikely ssDNA. to be This involved follows in because fork regression RecA is inhibitedwhere either by a the single-strand leading or bindinglagging strands protein (SSB)contain [38 exposed]. However, ssDNA. if DNA This damage follows is because present RecA in the is parental inhibited duplex by a single-strand DNA ahead of binding the fork, protein RecA (SSB)could [38]. nucleate However, nucleoprotein if DNA damage filaments is at present the site in of the damage, parental to duplex whichit DNA binds ahead with enhancedof the fork, a ffiRecAnity couldrelative nucleate to undamaged nucleoprotei DNAn [filaments40]. Filament at the extension site of damage, from
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  • Chromosome Duplication in Saccharomyces Cerevisiae

    Chromosome Duplication in Saccharomyces Cerevisiae

    | YEASTBOOK GENOME ORGANIZATION AND INTEGRITY Chromosome Duplication in Saccharomyces cerevisiae Stephen P. Bell*,1 and Karim Labib†,1 *Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and yMedical Research Council Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, DD1 5EH, United Kingdom ORCID ID: 0000-0002-2876-610X (S.P.B.) ABSTRACT The accurate and complete replication of genomic DNA is essential for all life. In eukaryotic cells, the assembly of the multi-enzyme replisomes that perform replication is divided into stages that occur at distinct phases of the cell cycle. Replicative DNA helicases are loaded around origins of DNA replication exclusively during G1 phase. The loaded helicases are then activated during S phase and associate with the replicative DNA polymerases and other accessory proteins. The function of the resulting replisomes is monitored by checkpoint proteins that protect arrested replisomes and inhibit new initiation when replication is inhibited. The replisome also coordinates nucleosome disassembly, assembly, and the establishment of sister chromatid cohesion. Finally, when two replisomes converge they are disassembled. Studies in Saccharomyces cerevisiae have led the way in our understanding of these processes. Here, we review our increasingly molecular understanding of these events and their regulation. KEYWORDS DNA replication; cell cycle; chromatin; chromosome duplication; genome stability;