Crystal structures of the BsPif1 helicase reveal that a major movement of the 2B SH3 domain is required for DNA unwinding

Abstract

Pif1 helicases are ubiquitous members of the SF1B family and are essential for maintaining genome stability. It was speculated that Pif1-specific motifs may fold in specific structures, conferring distinct activities upon it. Here, we report the crystal structures of the Pif1 helicase from Bacteroides spp with and without adenosine triphosphate (ATP) analog/ssDNA. BsPif1 shares structural similarities with RecD2 and Dda helicases but has specific features in the 1B and 2B domains. The highly conserved Pif1 family specific sequence motif interacts with and constraints a putative pin-loop in domain 1B in a precise conformation. More importantly, we found that the 2B domain which contains a specific extended hairpin undergoes a significant rotation and/or movement upon ATP and DNA binding, which is absolutely required for DNA unwinding. We therefore propose a mechanism for DNA unwinding in which the 2B domain plays a predominant role. The fact that the conformational change regulates Pif1 activity may provide insight into the puzzling observation that Pif1 becomes highly processive during break-induced replication in association with Polδ, while the isolated Pif1 has low processivity.

Structure and function of Pif1 helicase

“…Similar stacking interactions were observed between F98 in the 1B (pin) domain of Dda and the DNA [35]. Characterization of several Pif1 variants with single amino acid substitutions in the ATPase and DNA binding sites [32,33] confirms the roles previously established for these motifs based on studies of other helicases [37]. The DNA in the structure of BaPif1 with bound DNA and nucleotide makes a 90° turn as it enters the DNA binding site [33].…”

“…In both the BsPif1 and BaPif1 structures, the proteins contact the 6 nucleotides visible in the structures through stacking interactions with the bases and hydrogen bonding and electrostatic interactions with the backbone. F75 and P74 in the 1B domain (pin-loop or wedge domain) of both BaPif1 and BsPif1 stack with bases of the DNA [32,33]. Similar stacking interactions were observed between F98 in the 1B (pin) domain of Dda and the DNA [35].…”

“…The crystal structures of BsPif1 from Bacteroides spp [32], BaPif1 from Bacteroides sp. 2-1-16 [33], and the human PIF1 helicase domain (hPIF1-HD) [33] have been reported (Figure 1).…”

“…Residues in motifs I, III, IV, and VI contact the ATP in the BsPif1 and BaPif1 structures [32,33]. In BsPif1, Q145 from motif IV (and the analogous Q499 in RecD2) have been proposed to act as sensors to detect the presence or absence of the γ-phosphate [32,34]. The DNA (yellow) passes between the RecA-like domains and the 2B domain.…”

“…As has been observed with other helicases, binding of ATP and ssDNA results in large conformational changes in both BsPif1 and BaPif1 with the 2A and 2B domains rotating and shifting relative to the 1A domain. Introduction of a disulfide bond that prevents this rotation in BsPif1 or introduction of proline residues in the hinge between the 2A and 2B domains of BaPif1 abolishes dsDNA unwinding activity [32,33]. …”