Structure of the human RECQ1 helicase reveals a putative strand-separation pin

Abstract

RecQ-like helicases, which include 5 members in the human genome, are important in maintaining genome integrity. We present a crystal structure of a truncated form of the human RECQ1 protein with Mg-ADP. The truncated protein is active in DNA fork unwinding but lacks other activities of the full-length enzyme: disruption of Holliday junctions and DNA strand annealing. The structure of human RECQ1 resembles that of Escherichia coli RecQ, with some important differences. All structural domains are conserved, including the 2 RecA-like domains and the RecQ-specific zinc-binding and winged-helix (WH) domains. However, the WH domain is positioned at a different orientation from that of the E. coli enzyme. We identify a prominent beta-hairpin of the WH domain as essential for DNA strand separation, which may be analogous to DNA strand-separation features of other DNA helicases. This hairpin is significantly shorter in the E. coli enzyme and is not required for its helicase activity, suggesting that there are significant differences between the modes of action of RecQ family members.

Fig. 1.

Characterization of the RECQ1^T1 mutant. (A) Size exclusion chromatography profiles of RECQ1^FL (black) RECQ1^T1 (red), RECQ1^T1 + ATPγS (green), RECQ1^T1 + ATPγS + ssDNA (blue), RECQ1^T1 +ssDNA (purple). (B) Plot of DNA unwinding activity as a function of protein concentration using a fork-duplex substrate of 20 bp with ssDNA tails of 30 nt. Concentration dependence experiments were performed by using 0–100 nM RECQ1^FL (▵) or RECQ1^T1 (•). All of the reactions were stopped after 15 min. Data points were the mean of 3 independent experiments with the standard deviation indicated by error bars. The Inset shows an example of the unwinding assay using 0–200 nM RECQ1^T1. (C) Plot of the strand annealing activity as a function of protein concentration using the same partially complementary synthetic oligonucleotides used to prepare the forked duplex substrate. Concentration dependence experiments were performed by using 0–200 nM full-length RECQ1 (▵) or RECQ1^T1 (•). All of the reactions were stopped after 20 min. Data points were the mean of 3 independent experiments with the standard deviation indicated by error bars. The Inset shows an example of the strand annealing assay using 0–200 nM RECQ1^T1. (D) Concentration dependence experiments using 0–200 nM RECQ1^FL or RECQ1^T1 and a 50-bp-long synthetic HJ substrate that contains a 12-bp homologous core. All of the reactions were stopped after 20 min.

Fig. 2.

Overview of RECQ1 structure. (A) Ribbon representation of a single RECQ1 molecule, viewed from 3 perpendicular orientations. The subdomains are identified by color: Core helicase domain D1, red; core helicase domain D2, blue; zinc motif (ZnD), yellow; helical hairpin (HH), orange; WH domain, green; and the β-hairpin in purple. ADP is shown in space-filling form. (B) E. coli RecQ, colored as in A. [PDB ID code 1OYY (30)] The molecule is viewed in the same orientation as the central view in A, using the D2 domain as a reference. (C) Side-by-side comparison of bacterial RecQ (Left) and human RECQ1 (Right). Arrows indicate the rotations of the various domains. The WH domain rotates 90° around the vertical (compare orientation of helix α1 marked by asterisk), the HH tilts up by 10°, and the D1 rotates away from D2.

Fig. 3.

Details of the ADP and Zn-binding regions. (A) The nucleotide-binding pocket. Main chain and carbons are colored according the conserved motifs: motif 0 (yellow), motif I (magenta), motif II (light blue), motif V (gray). (B) Overlay of the Zn domains of RECQ1 (orange) and E. coli RecQ (gray). The yellow/black spheres indicate the zinc ion, which nearly overlap in the 2 structures. (C) Overlay of the WH domains of RECQ1 (red), WRN (blue), and E. coli RecQ (green). The β-hairpin forming one of the wings and the hydrophobic residues at the hairpin tip are highlighted.

Fig. 4.

Comparison of RECQ1 with DNA-bound structures of DNA helicases PcrA and HEL308. (A) PcrA (PDB ID code 3PJR). (B) HEL308 (PDB ID code 2P6R). (C) RECQ1, with DNA overlaid at the same relative positions as the experimentally determined DNA cocomplexes. The core helicase domains of the 3 proteins (depicted in red and blue) are presented in the same orientation; the DNA is depicted in black/purple, and a β-hairpin (magenta) near the point of DNA strand separation (marked by asterisk) is highlighted by green circles.

Fig. 5.

Characterization of the β-hairpin mutants of RECQ1 and E. coli RecQ. The mutant sequences are listed in Table 1. (A) Plot of the DNA fork unwinding activity as a function of RECQ1 concentration. Concentration-dependence experiments were performed by using the mutants indicated in the figure (2, 5, 10, 20, 40, 50, 80, 100, 150, 200 nM protein). All of the reactions were stopped after 15 min. Data points were the mean of 3 independent experiments with the standard deviation indicated by error bars. (B) Plot of the unwinding activity as a function of E. coli RecQ concentration. Concentration-dependence experiments were performed by using the RecQ mutants indicated in the figure (0.005, 0.01, 0.02, 0.05, 0.0625, 0.125, 0.25, 0.5, 2, 5, 10, 20 nM; only the low concentration range is shown). All of the reactions were stopped after 15 min. Data points were the mean of 3 independent experiments with the standard deviation indicated by error bars.

Fig. 6.

Alignment of the putative β-hairpins of RecQ family helicases. The sequences are arranged in 4 clusters according to the overall similarity in the entire RecQ catalytic region (PFAM domains DEAD, HelicC, RQC, and HRDC); residues are colored according to similarity within a subgroup or across the family. An aromatic residue, thought to be near the tip of the hairpin, is marked in purple; note that some of the BLM proteins lack such a residue. Sequences 12–14 are bacterial RecQ enzymes. The β-hairpin is significantly shorter that that in the eukaryotic enzymes. The vast majority of known bacterial RecQ sequences have a histidine (red) in place of an aromatic residue, as in E. coli. Two bacterial sequences shown have a phenylalanine in that position, indicating that the histidine may have a similar role to that of the aromatic residue in other RecQ helicases.