RecQ DNA helicases are critical enzymes for the maintenance of genome integrity. activity or nucleotide occupancy to ssDNA binding. The interaction of the ARL with the DNA is also observed in LY 344864 structures of DNA complexes of both RecQ [PDB ID code 4TMU (21)] and human BLM (PDB ID code 4CGZ). A structure-based sequence alignment of the RecA domains of SF2 helicases is shown LY 344864 in Fig. S3. The DNA-contacting residues of RECQ1 in motifs IV IVa and V have homologous counterparts in all SF2 proteins for which information is available. The contacts with the ARL are conserved uniquely in RecQ-family protein. DNA contacts with residues in motifs Ia and Ib are absent in our RECQ1 structure because the 3′ ssDNA does not extend into this region. ssDNA/dsDNA Junction. The last base pair of the duplex region in the crystal involves C21 of the top strand and a Gua base from the 5′ single-strand end of a DNA molecule from a neighboring molecule in the crystal (Fig. 2and Fig. S1shows the β-hairpin (purple) interacting with the D1 domain of the dimeric partner RECQ1 molecule (gray). The structure clearly shows that the dimerization and DNA-unwinding functions occur at opposite sides of the β-hairpin. Furthermore it suggests the possibility that the observed dimeric arrangement enhances helicase activity by buttressing the hairpin in a favorable orientation for strand separation. Fig. 2. Interactions with the dsDNA and branch point. (and and Table S1). These results provide the first glimpse to our knowledge of the RECQ1 mechanism of strand annealing. If the energy barrier to spontaneous annealing at room temperature is electrostatic repulsion and the loss of entropy when bringing two strands to proximity the protein may serve as a structural template to counteract the electrostatic repulsion and to nucleate a double-helical structure which can then propagate with little additional energetic cost. HJs Are Preferentially Bound by a Flat RECQ1 Tetramer. RECQ1FL protein possesses HJ branch migration activity (29 30 To gain insight into the mechanism of HJ recognition we tested if the T511 K514 and R528 mutations in the WH domain affect the branch migration activity of RECQ1. Our results showed that all these mutants have a reduced branch migration activity suggesting that there are indeed unique structural features that account for the HJ resolution activities of the five human RecQ proteins (Fig. 3 and Fig. S5(PDB ID code 4TMU) (21). That structure maintains the highly conserved ratchet structure around the ssDNA close to the branch point: Specifically residues R324 A346 T371 and K393 in RECQ1 are matched by the equivalent R246 A268 T293 and R315 in RecQ (CsRecQ). In addition the intercalating M429 is replaced by W424 (one α-helical turn away) in the bacterial structure. Crucially COL5A1 the ssDNA chain binds tightly to the ARL immediately next to the D2 contact followed by tight binding to helicase motifs Ia and Ib. Although this structure does not contain ATP Manthei et al. (21) argue that it represents a conformation poised for ATP hydrolysis. Thus it seems that the two structures represent two extreme states of the catalytic cycle. The translocation steps remain to be elucidated as does the timing of ATP hydrolysis and release. A recurrent feature of DNA helicases is the presence of a β-hairpin structure located at the junction of dsDNA and ssDNA; these structures have been shown previously by mutational (13) and structural analysis (17 22 to be essential for coupling ATPase LY 344864 activity to strand separation. As predicted the current structure shows a similar arrangement whereby the β-hairpin of the WH domain is in a LY 344864 position to affect strand separation both as a physical barrier and by providing an aromatic residue that may compensate for the loss of stacking at the last base pair of the DNA duplex. The WH domain (and the β-hairpin) is highly conserved structurally although the sequences and charge distribution vary considerably among different RecQ helicases (Fig. S8). This sequence variation in addition to changes in the spatial relationship LY 344864 between the WH domain and the remainder of the protein may have significant implications on substrate.