Human Pif1 helicase is a G-quadruplex DNA-binding protein with G-quadruplex DNA-unwinding activity

Abstract

Pif1 proteins are helicases that in yeast are implicated in the maintenance of genome stability. One activity of Saccharomyces cerevisiae Pif1 is to stabilize DNA sequences that could otherwise form deleterious G4 (G-quadruplex) structures by acting as a G4 resolvase. The present study shows that human Pif1 (hPif1, nuclear form) is a G4 DNA-binding and resolvase protein and that these activities are properties of the conserved helicase domain (amino acids 206-620 of 641, hPifHD). hPif1 preferentially bound synthetic G4 DNA relative to ssDNA (single-stranded DNA), dsDNA (double-stranded DNA) and a partially single-stranded duplex DNA helicase substrate. G4 DNA unwinding, but not binding, required an extended (>10 nucleotide) 5' ssDNA tail, and in competition assays, G4 DNA was an ineffective suppressor of helicase activity compared with ssDNA. These results suggest a distinction between the determinants of G4 DNA binding and the ssDNA interactions required for helicase action and that hPif1 may act on G4 substrates by binding alone or as a resolvase. Human Pif1 could therefore have a role in processing G4 structures that arise in the single-stranded nucleic acid intermediates formed during DNA replication and gene expression.

Suppression of Apoptosis by PIF1 Helicase in Human Tumor Cells

“…Thus, it was proposed that PIF1 function is redundant in mammalian cells. Nevertheless, in vitro analysis of purified full-length PIF1 suggested that it bound and unwound DNA structures that might form during DNA replication stress, including structures resembling stalled replication forks (14) or G-quadruplexes (28). The effects of PIF1 depletion on S-phase transition, checkpoint activation, and apoptosis in tumor cells are consistent with these in vitro results; however, nontumor cell lines did not show similar responses.…”

G-quadruplexes Significantly Stimulate Pif1 Helicase-catalyzed Duplex DNA Unwinding

“…Experimental Design for Kinetic Characterization of Pif1-mediated G-quadruplex DNA Unwinding-Pif1 was reported previously as a low or nonprocessive helicase for unwinding duplex DNA (19,38,46), but it is highly active for unwinding G4s (19,47). In fact, the G4s do not exist alone as an isolated motif, but instead they are usually connected to double-stranded DNA at one or both sides, such as the G4s implicated in stalled replication forks and in replication origins (Fig.…”

“…Previous studies have shown that cells have evolved mechanisms for regulating G4 structures to counteract their intrinsic recombinogenic effects. It has been shown that several helicases such as those in the RecQ (15), DEAH-box (16,17), and Pif families (18,19) possess G4 resolving activity.…”

DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase

“…ScRrm3 also promotes fork progression at other sites where ScPif1 is not known to act, such as at tRNA genes, inactive origins, and the silent mating type loci, while ScPif1 is important for replication through G-rich motifs that can form G-quadruplex structures in vitro (Ivessa et al 2003;Paeschke et al 2011). Human Pif1 (hPif1) also unwinds G-quadruplex structures in vitro, as well as DNA structures that resemble stalled replication forks (George et al 2009;Sanders 2010). Although S. cerevisiae Pif1 family helicases have important roles in DNA replication, pif1D rrm3D cells are viable (Ivessa et al 2000).…”