Replication stress induces specific enrichment of RECQ1 at common fragile sites FRA3B and FRA16D

Abstract

BackgroundStalled replication forks at common fragile sites are a major cause of genomic instability. RecQ helicases, a highly conserved family of DNA-unwinding enzymes, are believed to ease ‘roadblocks’ that pose challenge to replication fork progression. Among the five known RecQ homologs in humans, functions of RECQ1, the most abundant of all, are poorly understood. We previously determined that RECQ1 helicase preferentially binds and unwinds substrates that mimic DNA replication/repair intermediates, and interacts with proteins involved in DNA replication restart mechanisms.MethodWe have utilized chromatin immunoprecipitation followed by quantitative real-time PCR to investigate chromatin interactions of RECQ1 at defined genetic loci in the presence or absence of replication stress. We have also tested the sensitivity of RECQ1-depleted cells to aphidicolin induced replication stress.ResultsRECQ1 binds to the origins of replication in unperturbed cells. We now show that conditions of replication stress induce increased accumulation of RECQ1 at the lamin B2 origin in HeLa cells. Consistent with a role in promoting fork recovery or repair, RECQ1 is specifically enriched at two major fragile sites FRA3B and FRA16D where replication forks have stalled following aphidicolin treatment. RECQ1-depletion results in attenuated checkpoint activation in response to replication stress, increased sensitivity to aphidicolin and chromosomal instability.ConclusionsGiven a recent biochemical observation that RECQ1 catalyzes strand exchange on stalled replication fork structures in vitro, our results indicate that RECQ1 facilitates repair of stalled or collapsed replication forks and preserves genome integrity. Our findings provide the first evidence of a crucial role for RECQ1 at naturally occurring fork stalling sites and implicate RECQ1 in mechanisms underlying common fragile site instability in cancer.

Cellular Deficiency of Werner Syndrome Protein or RECQ1 Promotes Genotoxic Potential of Hydroquinone and Benzo[a]pyrene Exposure

“…Alternatively, or in addition, regressed fork may be cleaved by structure-specific nucleases to generate a DSB which is subsequently repaired by HR (Figure 4) 23 . Both RECQ1 and WRN are recruited at arrested replication forks 45 . Demonstrated preference of RECQ1, but not WRN, for fork reversal 13 supports increased DSBs in RECQ1-depleted cells exposed to BaP.…”

Human RecQ Helicases in DNA Repair, Recombination, and Replication

“…WRN specifically increases the ability of Pol δ to replicate through common fragile sites, such as FRA16D (207). RECQL1 also localizes to FRA16D and FRA3B in response to replicative stress (208) and may play a similar role in CFS DNA replication.…”

Site-directed mutants of human RECQ1 reveal functional importance of the zinc binding domain

“…Cells lacking RECQ1 have a higher rate of spontaneous sister chromatid exchange, are more sensitive to genotoxins, and undergo more double-stranded DNA breaks [8–12]. RECQ1 seems to prevent such DNA breaks by stabilizing stalled or regressed replication forks [13–15]. In addition, RECQ1 is involved in non-homologous end joining [16] and lengthening of telomeres without telomerase [17].…”