A Monomer of Pif1 Unwinds Double-Stranded DNA and It Is Regulated by the Nature of the Non-Translocating Strand at the 3′-End

Abstract

Using a DNA polymerase-coupled assay and FRET-based helicase assays, in this work we show that a monomer of S. cerevisiae Pif1 can unwind double-stranded DNA. The helicase activity of a Pif1 monomer is modulated by the nature of the 3′-ssDNA tail of the substrate and its effect on a Pif1-dependent re-winding activity that is coupled to the opening of dsDNA. We propose that in addition to the ssDNA site on the protein that interacts with the translocating strand, Pif1 has a second site that binds the 3′-ssDNA of the substrate. Interaction of DNA with this site modulates the degree to which re-winding counteracts unwinding. Depending on the nature of the 3′-tail and the length of the duplex DNA to be unwound this activity is sufficiently strong to mask the helicase activity of a monomer. In excess Pif1 over the DNA the Pif1-dependent re-winding of the opened DNA strongly limits unwinding, independent of the 3′-tail. We propose that in this case binding of DNA to the second site is precluded and modulation of the Pif1-dependent re-winding activity is largely lost.

Role of the Pif1-PCNA Complex in Pol δ-Dependent Strand Displacement DNA Synthesis and Break-Induced Replication

“…Importantly, even though stimulation of synthesis still occurred upon substitution of Pif1 with the R3E mutant, much shorter products were generated in the latter case (Figures 3Aii and 3Aiii, lanes 10–13). We attribute the stimulatory, albeit attenuated effect of the R3E mutant in DNA synthesis to its residual PCNA interaction capability (data not shown) or to a PCNA interaction-independent action of Pif1 on the DNA substrate (Pike et al, 2009; Singh et al, 2016). …”

“…However, it is unclear what other properties of Pif1 may be important for Pol δ-mediated repair DNA synthesis. Pif1 possesses a conserved helicase core, and its N-terminal region (amino acids 40–233) (Figure 1A) appears to be unstructured and is dispensable for the helicase function (Singh et al, 2016). The C-terminal region of Pif1 (amino acids 788–859) (Figure 1A) is a target of Mec1-Rad53-induced phosphorylation for the regulation of telomerase at DSBs (Makovets and Blackburn, 2009; Vasianovich et al, 2014).…”

Chemo-mechanical pushing of proteins along single-stranded DNA

“…EcRep can disrupt dsDNA binding of proteins such as lac repressor (39,40). ScPif1 is involved in displacing telomerase from telomeric repeats and double-strand breaks (41)(42)(43)(44). EcRecBCD can displace histones (45) and also push dsDNA bound proteins along dsDNA (46,47).…”

Structure and function of Pif1 helicase

“…For human PIF1, the N-terminal domain has been proposed to be involved in strand annealing [29]. Deletion of the C-terminal domain decreases the processivity of unwinding for ScPif1 [30]. Additionally, phosphorylation of the C-terminal domain of ScPif1 on T763 and S766 in response to DSBs is required for ScPif1 inhibition of telomerase at DSBs but not at telomeres [31].…”

“…Although monomeric forms of some helicases can function, [30,42–48] multimeric forms can provide multiple DNA binding sites which typically increases processivity and can provide additional avenues for regulation of activity. Dimeric helicases have been described in which the conformations of each respective monomer can activate or inhibit the enzyme [48,49].…”