RecQ and RecJ process blocked replication forks prior to the resumption of replication in UV-irradiated Escherichia coli

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The error-free repair of double-stranded DNA breaks by homologous recombination requires processing of broken ends. These processed ends are substrates for assembly of DNA strand exchange proteins that mediate DNA strand invasion. Here, we establish that human BLM helicase, a member of the RecQ family, stimulates the nucleolytic activity of human exonuclease 1 (hExo1), a 5 33 double-stranded DNA exonuclease. The stimulation is specific because other RecQ homologs fail to stimulate hExo1. Stimulation of DNA resection by hExo1 is independent of BLM helicase activity and is, instead, mediated by an interaction between the 2 proteins. Finally, we show that DNA ends resected by hExo1 and BLM are used by human Rad51, but not its yeast or bacterial counterparts, to promote homologous DNA pairing. This in vitro system recapitulates initial steps of homologous recombination and provides biochemical evidence for a role of BLM and Exo1 in the initiation of recombinational DNA repair.Bloom syndrome ͉ Rad51 ͉ recombination ͉ RecQ ͉ DNA pairing H omologous recombination contributes toward the maintenance of genomic integrity by accurately repairing doublestranded DNA (dsDNA) breaks. The recombinational repair of dsDNA breaks (DSBs) proceeds by successive reactions that start with processing of the broken DNA ends to reveal single-stranded DNA (ssDNA) (1). Processing requires the action of a helicase and/or nuclease. The resultant 3Ј-terminated ssDNA is used by a DNA strand exchange protein as the substrate for assembly of the nucleoprotein filament that is the active species in the search for homologous DNA and subsequent DNA pairing. It is unclear which enzymes in eukaryotes resect the DNA break to initiate recombination; however, much is known about the process in bacteria. In Escherichia coli, recombinational DNA repair occurs by either the RecBCD or the RecF pathway (1). RecBCD is a helicase/nuclease that processes dsDNA to produce 3Ј-tailed ssDNA onto which RecA is loaded. In the RecF pathway, a separate helicase and nuclease are used for resection of DSBs and ssDNA-gaps: RecQ, a 3Ј35Ј helicase, and RecJ, a 5Ј33Ј exonuclease (1).

Section: Discussionmentioning

confidence: 78%

mentioning

confidence: 99%

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confidence: 99%

See 1 more Smart Citation

Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair

Nimonkar

Özsoy

Genschel³

et al. 2008

269

236

“…At the initiation stage of DNA recombination, RecQ unwinds dsDNA and allows a RecJ, a 5′ to 3′ exonuclease to process the ssDNA, resulting in 3′ terminated overhang used for homologous pairing by the RecA protein (40). In addition, these two proteins also process ssDNA gaps at the sites of stalled replication forks (41). For homologous recombination, the minimal efficient processing segment (MEPS) is approximately 40 bp for the RecF-dependent pathway (42).…”

Section: Recq Couples the Hydrolysis Of One Atp Molecule To The Movemmentioning

confidence: 99%

Efficient coupling of ATP hydrolysis to translocation by RecQ helicase

Rad

Kowalczykowski

2012

Helicases are ubiquitous enzymes that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) during essential processes such as replication, transcription, or repair. The Escherichia coli RecQ protein is a 3′ to 5′ helicase, which functions in the processes of homologous recombination and replication fork restart. Here, we analyzed the relationship between ATP hydrolysis by RecQ and its translocation on ssDNA. We monitored a single round of RecQ translocation on ssDNA by measuring the rates of inorganic phosphate release during translocation, and the dissociation of RecQ from ssDNA. We find that RecQ translocates with a rate of 16ðAE4Þ nucleotides/s and moves on average only 36ðAE2Þ nucleotides before dissociating. Fitting to an n-step kinetic model suggests that the helicase displays a nonuniform translocation mechanism in which it moves approximately five nucleotides rapidly before undergoing a rate-limiting kinetic slow step. Unexpectedly, RecQ requires a length of 34ðAE3Þ nucleotides to bind and translocate on ssDNA. This large site size suggests that several monomers are required to bind DNA prior to translocation. Energetically, the RecQ helicase couples the hydrolysis of one ATP molecule to the translocation of more than one nucleotide (1.6 AE 0.3). Thus, our data show that RecQ translocates on ssDNA by efficiently coupling the hydrolysis of one ATP molecule into structural alterations that result in movement of approximately two nucleotides, presumably by an inchworm mechanism. These attributes are consistent with the function of RecQ in recombination and replication.DNA motor | DNA repair | DNA unwinding | recombination A diverse group of enzymes, the helicases play a prominent role in replication, repair, and recombination by unwinding double-stranded DNA (dsDNA). Moreover, these motor enzymes function by translocating on single-stranded DNA (ssDNA)-a process that can be as important in vivo as unwinding dsDNA. Important cellular processes that are carried out by translocating enzymes include removal of secondary structure from ssDNA, movement of Holliday junctions, and displacement of bound proteins from ssDNA (1).A 3′ to 5′ helicase from E. coli, RecQ is the founding member of the RecQ-family of helicases (2). These enzymes belong to helicase superfamily-2 (SF2), but are more closely homologous to one another (3-5). Members of this family include Bloom (BLM), Werner (WRN), RECQ1, RECQ4, and RECQ5 from humans, Sgs1 from Saccharomyces cerevisiae, and Rqh1 from Schizosaccharomyces pombe (6). These proteins play important roles in DNA recombination and repair in their respective organisms. The function of BLM, WRN, and RECQ4 proteins are of particular interest because mutations in these helicases lead to Bloom's, Werner's, and Rothmund-Thomson syndromes, respectively. These genetic disorders are characterized by genomic instability and an increased incidence of cancer (6).Recent studies of helicases are demonstrating the importance of translocation in lieu of, or in addition to, helicas...

“…coli RecQ is a 3′ → 5′ helicase that functions in DNA-break repair by homologous recombination (2,5,6). RecQ and RecJ, a 5′ → 3′ exonuclease, process ssDNA gaps or dsDNA breaks into ssDNA for recombinational repair by RecA (7)(8)(9)(10). In addition, RecQ ensures recombination fidelity in vivo by removing inappropriately paired joint molecules to prevent illegitimate recombination and also by disrupting joint molecule intermediates to facilitate repair by synthesis-dependent strand annealing, preventing chromosomal crossing over (7,9,11,12).…”

mentioning

confidence: 99%

Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

Rad

Forget

Baskin

et al. 2015

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40-60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.recombination | fluorescence | TIRF microscopy | DNA repair | BLM