Structural and Functional Insights into the Unwinding Mechanism of Bacteroides sp Pif1.

Xianglian Zhou,Chen Chen,Sakshibeedu R Bharath,Yang He,Haiwei Song,Dewang Li,Zhou Liu,Wendan Ren,Xuhua Tang

doi:10.1016/j.celrep.2016.02.008

Abstract

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 β hairpin in other SF1B DNA helicases folds into an extended loop followed by an α 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.

Highlights

The prototypical member of Pif1 family helicases was first identified from mitochondria of Saccharomyces cerevisiae (ScPif1) (Lahaye et al, 1993)
Fluorescence resonance energy transfer experiments showed that a ScPif1 monomer is preferentially recruited to 30 single-stranded DNAs (ssDNAs)/ double-stranded DNAs (dsDNAs) junctions and induces repetitive DNA looping that is tightly coupled to its translocation activity powered by its ATPase (Zhou et al, 2014)
Given that subsequent efforts to crystallize Human Pif1 (hPif1) in complex with DNA substrates failed, we focused on Bacteroides sp Pif1 (BaPif1), which shares a sequence identity of 29% with hPif1-HD (Figure S1)

Summary

Graphical Abstract

Bharath, ..., Zhou Liu, Dewang Li, Haiwei Song. Zhou et al report the crystal structures of the helicase core domain of human Pif as well as Bacteroides sp Pif in different conformational states. The results reveal the functional role of the Pif signature motif and provide mechanistic insights into the unwinding activity of the Pif family helicases. Highlights d Structures of Bacteroides sp Pif in different conformational states are reported d The wedge region folds into an extended loop followed by an a helix.

SUMMARY

INTRODUCTION

RESULTS AND DISCUSSION

Conclusions

EXPERIMENTAL PROCEDURES