Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: Insights from DNA complex structures

Abstract

RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration.DNA helicases | RecQ | genome stability | Holliday junction | fork reversal R ecQ helicases are a family of ATP-dependent motor proteins that play central roles in maintaining genome stability. Defects in three of the five human RecQ homologs give rise to distinct genetic disorders associated with genomic instability, cancer predisposition, and premature aging (1-5). The unique clinical features of these disorders support the notion that the different RecQ helicases have nonoverlapping functions, but the molecular basis for their different enzymatic activities remains unclear. RecQ helicases catalyze ATP-dependent DNA unwinding in the 3′-5′ direction. Additionally, members of this helicase family have been shown to tackle an unparalleled breath of noncanonical DNA structures, such as fork DNA, G-quadruplexes, D-loops, and Holliday junction (HJ) structures (6-8). However, our understanding of the physical mechanisms by which RecQ helicases recognize and process their physiological substrates remains remarkably limited.RECQ1 is the shortest of the human RecQ-family helicases, comprising the bipartite ATPase domain common to all superfamily 2 (SF2) helicases, the RecQ-specific C-terminal domain (RQC), and short extensions on the N and C termini. We recently discovered a specific function of RECQ1 in branch migration and restart of reversed DNA replication forks upon DNA topoisomerase I inhibition that is not shared by other human RecQ helicases, such as Werner (WRN) or Bloom (BLM) syndrome proteins (9). On the other hand, BLM is the sole human RecQ helicase member specifically able to resolve double-HJ junction structures in conjunction with DNA topoisomerase III alpha and the RMI1 and RMI2 accessory proteins (10-12). These findings lead us to hypothesize that the sp...

A Monomer of Pif1 Unwinds Double-Stranded DNA and It Is Regulated by the Nature of the Non-Translocating Strand at the 3′-End

“…Second, the unwinding activity of a helicase could be underestimated if the protein can catalyze re-annealing of the substrate that is being unwound. The ability of Pif1 and other helicases to catalyze annealing of complementary ssDNA has been documented 32,42–49 . Therefore, we designed an assay that should bypass these two complications in determining the unwinding ability of a helicase.…”

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

“…RECQ1, however, lacks HRDC domain present in several RecQ proteins including bacterial RecQ and human WRN and BLM proteins [19]. Biochemically, RECQ1 participates in the processing of DNA structure intermediates of replication and repair by catalyzing ATP-dependent 3’–5’ unwinding of a variety of duplex DNA substrates and promoting branch migration of Holliday junction and D-loops[7,20–22]. Moreover, RECQ1 supports base-pairing of complementary single strand DNA in an ATP-independent manner [7,20–22].…”

The Human RecQ4 Helicase Contains a Functional RecQ C-terminal Region (RQC) That Is Essential for Activity

“…There is some discrepancy as to the role of the ␤-hairpin in the annealing activity: whereas mutation of the aromatic residue of RecQ4 does abolish annealing (Fig. 2H), the same mutation in RecQ1 does not (20). However, the annealing activity in RecQ1 appears to be subjected to a complex regulation, requiring the full-length protein and the formation of a tetrameric assembly, whereas the monomeric truncated form of RecQ4 here described exhibits annealing activity.…”

“…2A), suggesting the presence of complexes with different stoichiometries. These results could be explained either by a second RecQ4 helicase binding to the DNA substrate or by the formation of a 2:2 protein-DNA complex, as seen in the crystal structure of RecQ1 (20). Although we cannot rule FIGURE 1.…”