Xianglian Zhou1,2,4, Wendan Ren1,2,4, Sakshibeedu R. Bharath2,4, Xuhua Tang2, Yang He1, Chen Chen2, Zhou Liu2, Dewang Li1 and Haiwei Song1,2,3*
1 Life Sciences Institute and Innovation Center for Cell Signaling Network, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, China
2 Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Singapore
3 Department of Biochemistry, National University of Singapore, 14 Science Drive, Singapore 117543, Singapore
4 Co-first author
Published in Cell Reports on 1 March 2016.
Pif1 is a conserved SF1B DNA helicase involved in maintaining genome stability through unwinding double-stranded DNAs (dsDNAs), DNA/RNA hybrids, and G quadruplex (G4) structures. Here, we report the structures of the helicase domain of human Pif1 and Bacteroides sp Pif1 (BaPif1) in complex with ADP-AlF4– and two different single-stranded DNAs (ssDNAs). The wedge region equivalent to the b hairpin in other SF1B DNA helicases folds into an extended loop followed by an a helix. The Pif1 signature motif of BaPif1 interacts with the wedge region and a short helix in order to stabilize these ssDNA binding elements, therefore indirectly exerting its functional role. Domain 2B of BaPif1 undergoes a large conformational change upon concomitant binding of ATP and ssDNA, which is critical for Pif1’s activities. BaPif1 cocrystallized with a tailed dsDNA and ADP-AlF4–, resulting in a bound ssDNA bent nearly 90° at the ssDNA/dsDNA junction. The conformational snapshots of BaPif1 provide insights into the mechanism governing the helicase activity of Pif1.
Figure legend: Insights into DNA Unwinding Activity of BaPif1
(A) Cartoon representation of BaPif1 with the modeled duplex DNA (yellow). Nucleotides C9 and G10 observed in BaPif1-dH are superposed with nucleotides 2 and 3 of the modeled duplex to represent the bent conformation of the duplex region. As a result of translocation and bending of phosphate backbone,
it is likely that the first two base pairs of the duplex region are unwound, as represented in the model. The wedge and the signature motif are also shown.
(B) Proposed mechanism of duplex unwinding by BaPif1.
In the apo form, the accessory domain 2B in BaPif1 is in an inhibitory conformation so that the ssDNA binding site is occluded. The concomitant binding of tailed dsDNA and ATP results in rotation of domains 2A and 2B relative to domain 1A. Translocation of the Pif1 helicase would lead to a duplex region clash with the wedge region. Such steric hindrance imposed by the wedge region with the incoming duplex would induce the bending of the phosphate backbone of the DNA duplex, as evidenced in BaPif1-dH. Subsequently, the Pif1 helicase would restore to the apo form after complete unwinding of the DNA duplex.
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