Structure of the helicase core of Werner helicase, a key target in microsatellite instability cancers

WRN expression and DNA damage in engineered cell lines. (A) To monitor cellular expression and nuclear accumulation of the WRNr transgenes, an anti-FLAG immunofluorescence analysis was performed in monoclonal cell lines expressing wild-type and mutant variants of WRNr. Scale bar, 20 μM. (B) Representative images of the immunofluorescence analysis of γ-H2AX that was performed 72 h after siRNA transfection. Scale bar, 20 μM. (C) Mitotic chromosome spread analysis was performed on each of the transgenic cell lines 72 h post-siRNA transfection that included a 6 h nocodazole (1.5 μM) treatment. Quantification was performed under a microscope where each mitotic spread was categorized into less than five (<5) breaks or more than five (>5) breaks (n ≥ 28 mitotic spreads). Data values and error bars presented here are the mean and the SD, respectively, from biological repeats (n = 2).

Details of nucleotide binding and comparisons with other human RecQ helicases. (A) Comparison of the nucleotide binding modes of WRN (left), RECQL5 (centre), and BLM (right) helicases. The nucleotide in the WRN structure is bound with extensive contacts from motif VI, whereas contacts from motif I are less extensive than in other RecQ helicase structures. (B) WRN electron density maps, the final 2F_o-1F_c electron density map is shown in grey in the vicinity of the nucleotide-binding site contoured at 1.2σ. A F_o-F_c difference map is show in green contoured at 2.7σ. This map was calculated with the side chain of R857 omitted from the model used for map calculation. (C) Close-up view of the WRN-specific β-hairpin insertion within the D2 domain, the hydrogen bond formed from the type II′ β-turn is show in yellow dashes.

The upper panel shows a structural superposition of selected RecQ family structures on the basis of the D1 domain (left) or D2 domain (right). Invariant points used to define the positioning of the two domains are depicted as red spheres with numbering according to the WRN sequence. The lower panel shows a 2D vector diagram of distances between the vector pairs. The WRN structure occupies a distinct position at the bottom of the plot with a conformation even more compact that the APO form structures.

The double-stranded part of the DNA is positioned variably with respect to the D2 domain, whereas the single-stranded 3′ overhang can be seen to converge an makes conserved interactions with helicase motifs IV and V.