Catalytic Strand Separation by RECQ1 Is Required for RPA-Mediated Response to Replication Stress

Abstract

Three (BLM, WRN, and RECQ4) of the five human RecQ helicases are linked to genetic disorders characterized by genomic instability, cancer, and accelerated aging [1]. RECQ1, the first human RecQ helicase discovered [2-4] and the most abundant [5], was recently implicated in breast cancer [6, 7]. RECQ1 is an ATP-dependent DNA-unwinding enzyme (helicase) [8, 9] with roles in replication [10-12] and DNA repair [13-16]. RECQ1 is highly expressed in various tumors and cancer cell lines (for review, see [17]), and its suppression reduces cancer cell proliferation [14], suggesting a target for anti-cancer drugs. RECQ1's assembly state plays a critical role in modulating its helicase, branch migration (BM), or strand annealing [18, 19]. The crystal structure of truncated RECQ1 [20, 21] resembles that of E.coli RecQ [22] with two RecA-like domains, a RecQ-specific zinc-binding domain and a winged-helix domain, the latter implicated in DNA strand separation and oligomer formation. In addition, a conserved aromatic loop (AL) is important for DNA unwinding by bacterial RecQ [23, 24] and truncated RECQ1 helicases [21]. To better understand the roles of RECQ1, two AL mutants (W227A and F231A) in full-length RECQ1 were characterized biochemically and genetically. The RECQ1 mutants were defective in helicase or BM but retained DNA binding, oligomerization, ATPase, and strand annealing. RECQ1-depleted HeLa cells expressing either AL mutant displayed reduced replication tract length, elevated dormant origin firing, and increased double-strand breaks that could be suppressed by exogenously expressed replication protein A (RPA). Thus, RECQ1 governs RPA's availability in order to maintain normal replication dynamics, suppress DNA damage, and preserve genome homeostasis.