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

Abstract

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 (RECQ149–616) shows a prominent β-hairpin, with an aromatic residue (Y564) at the tip, located in the C-terminal winged-helix domain. Here, we show that the β-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 β-hairpin is required for dimer formation in RECQ149–616 and tetramer formation in full-length RECQ1. We confirmed the presence of stable RECQ149–616 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 β-hairpin and the other the N-terminus of RECQ1, suggesting a non-hierarchical mechanism of tetramer assembly.

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

“…1B). This finding is compatible with the observation that the dimer is not essential for fork unwinding activity, and that dimer formation is not dependent on DNA (16). In the description below, we follow the trajectory of the DNA along a RECQ1 monomer, and explore the implications for the mechanism of helicase action.…”

“…1A). Mutational analysis has demonstrated that the dimeric structure is stimulatory but not essential for DNA unwinding activity of RECQ1 T1 ; this observation contrasts with the essential requirement for the β-hairpin structure (13,16).…”

“…Full-length RECQ1 purifies as a mixture of tetramers and dimers (16). The truncated version of the protein used in this study (RECQ1 T1 ) lacks the N terminus, which is required for tetramerization; the protein appears as a dimer both in solution and in crystals.…”

“…The truncated version of the protein used in this study (RECQ1 T1 ) lacks the N terminus, which is required for tetramerization; the protein appears as a dimer both in solution and in crystals. The dimer is arranged head to tail, with the β-hairpin in each WH domain interacting with the N-terminal helicase domain (D1) of the opposite subunit (16) (Fig. 1A).…”

“…The top strand, which follows a normal helical trajectory up to this point, is sharply kinked so that the next base (T 22 ) is unstacked and points away from the helix axis, into a wide groove that could accommodate either a purine or pyrimidine base. We have shown before that the β-hairpin, in addition to its role in DNA strand separation, also forms part of the contact surface of the RECQ1 dimer (16); mutations that selectively disrupt the protein/protein surface did not abolish DNA unwinding but reduced its efficiency by 60-80%. Fig.…”