A prominent b-hairpin structure in the winged-helix domain of RECQ1 is required for DNA unwinding and oligomer formation

RecQ helicases have attracted considerable interest in recent years due to their role in the suppression of genome instability and human diseases. These atypical helicases exert their function by resolving a number of highly specific DNA structures. The crystal structure of a truncated catalytic core of the human RECQ1 helicase (RECQ1) shows a prominent b-hairpin, with an aromatic residue (Y564) at the tip, located in the C-terminal winged-helix domain. Here, we show that the b-hairpin is required for the DNA unwinding and Holliday junction (HJ) resolution activity of full-length RECQ1, confirming that it represents an important determinant for the distinct substrate specificity of the five human RecQ helicases. In addition, we found that the b-hairpin is required for dimer formation in RECQ1 and tetramer formation in full-length RECQ1. We confirmed the presence of stable RECQ1 dimers in solution and demonstrated that dimer formation favours DNA unwinding; even though RECQ1 monomers are still active. Tetramers are instead necessary for more specialized activities such as HJ resolution and strand annealing. Interestingly, two independent protein–protein contacts are required for tetramer formation, one involves the b-hairpin and the other the N-terminus of RECQ1, suggesting a nonhierarchical mechanism of tetramer assembly. INTRODUCTION Since the discovery of the first RecQ enzyme from Escherichia coli over 20 years ago (1), numerous other RecQ family helicase genes have been found in various organisms ranging from prokaryotes to eukaryotes. Unicellular organisms, such as bacteria and yeast have only one or two RecQ helicase genes per species, while higher eukaryotes generally express multiple RecQ enzymes (2–4). Five members of the RecQ family have been found in human cells: BLM, RECQ1 (alias RECQL or RECQL1), RECQ4 (RECQL4), RECQ5 (RECQL5) and WRN. Mutations in the genes encoding BLM, WRN and RECQ4 are linked to defined genetic disorders associated with genomic instability, cancer predisposition and features of premature aging (5–9). No heritable cancer predisposition disorder has been associated with mutations in the remaining two human RecQ helicase genes, RECQ1 and RECQ5 as yet. However, recent studies have linked a single-nucleotide polymorphism present in the RECQ1 gene to a reduced survival in pancreatic cancer patients (10). Recent reports showing that RECQ1 silencing has anti-cancer effect both in cellular and mouse xenograft models suggest that RECQ1 may have a role in cancer survival, and might be considered as a new suitable target for cancer therapy (11–13). RecQ helicases unwind DNA with a 30–50 polarity and are capable of unwinding a variety of DNA structures in addition to standard B-form DNA duplexes, i.e. triple helices, 3or 4-way junctions, and G-quadruplex DNA (14–17). Consistent with an ability to unwind various DNA structures, several cellular functions have been *To whom correspondence should be addressed. Tel: +39 040 3757326; Fax: +39 04