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.
Highlights
Stalled replication forks at common fragile sites are a major cause of genomic instability
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
To test whether RECQ1 plays a role in mammalian fork progression after initiation, we investigated the effects of fork stalling on binding of RECQ1 to lamin B2 origin in HeLa cells by chromatin immunoprecipitation (ChIP) (Figure 1B, C)
Summary
Stalled replication forks at common fragile sites are a major cause of genomic instability. Common fragile sites (CFS) are specific chromosomal regions, extending over large DNA sequences, which are especially prone to genomic instability. Members of RecQ family DNA helicases, represented by five homologs (BLM, WRN, RecQ4, RecQ5β and RECQ1) in humans, are implicated in genome maintenance through repair of stalled or collapsed replication forks [8,9]. RecQ helicases are recruited to the sites of arrested DNA replication where in addition to serving repair functions, they help stabilize stalled replication complexes and likely contribute to the S-phase checkpoints [10,11,12,13,14]. Functions of RecQ helicases are likely to be critical under replication stress; among these, the roles of WRN and BLM remain best characterized while there is only limited information available for other RecQ proteins
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