Molecular mechanism of G-quadruplex unwinding helicase: sequential and repetitive unfolding of G-quadruplex by Pif1 helicase

Abstract

Recent advances in G-quadruplex (G4) studies have confirmed that G4 structures exist in living cells and may have detrimental effects on various DNA transactions. How helicases resolve G4, however, has just begun to be studied and remains largely unknown. In the present paper, we use single-molecule fluorescence assays to probe Pif1-catalysed unfolding of G4 in a DNA construct resembling an ongoing synthesis of lagging strand stalled by G4. Strikingly, Pif1 unfolds and then halts at the ss/dsDNA junction, followed by rapid reformation of G4 and 'acrobatic' re-initiation of unfolding by the same monomer. Thus, Pif1 unfolds single G4 structures repetitively. Furthermore, it is found that Pif1 unfolds G4 sequentially in two large steps. Our study has revealed that, as a stable intermediate, G-triplex (G3) plays an essential role in this process. The repetitive unfolding activity may facilitate Pif1 disrupting the continuously reforming obstructive G4 structures to rescue a stalled replication fork. The proposed mechanism for step-wise unfolding of G4 is probably applicable to other helicases that resolve G4 structures for maintaining genome stability.

Structure and function of Pif1 helicase

“…The consensus is that folding of G4DNA structures proceeds slowly to the thermodynamic equilibrium; however, fast folding to various intermediate occurs [84,85]. These reported folding rates differ from those reported in smFRET studies, which indicate very rapid folding [61,86]. It is possible that that the rapid conformational changes observed in smFRET are between unfolded and intermediate states or the fully folded and intermediates states as opposed to fully folded and fully unfolded states.…”

“…ScPif1 has also been described as having patrolling activity in which it binds to a 3′-ssDNA tailed dsDNA junction and repetitively reels in the tail in single nucleotide steps [61,86] (Figure 4C). Because the helicase remains anchored at the 3′-ssDNA junction, a loop of ssDNA is created as the helicase reels in the tail as it translocates toward the 3′-end of the DNA.…”

“…This action is proposed to occur repetitively (about 200 times to unwind a 31 base pair DNA:RNA hybrid duplex [61]). Multiple groups found that G4DNA was unfolded in each patrolling cycle, but dsDNA downstream of the G4DNA was not melted which suggested a mechanism whereby ScPif1 repetitively unfolds the G4DNA which refolds rapidly but leaves adjacent dsDNA intact [61,86]. However, other groups have reported that ScPif1 is able to unwind a duplex following a quadruplex [68,82,83].…”

“…The differences are likely due to the substrate design and experimental conditions because some reports used non-forked duplexes [61,68,83,86] which are known to be non-preferred substrates for ScPif1 while another used both forked and non-forked duplexes [82]. Some experiments were performed under single-cycle conditions [61,68,82,86] and others under multiple turnover conditions [82,83]. Some used excess enzyme [68,82] and others used excess substrate [61,83,86].…”

“…Some experiments were performed under single-cycle conditions [61,68,82,86] and others under multiple turnover conditions [82,83]. Some used excess enzyme [68,82] and others used excess substrate [61,83,86]. It is likely that the differences in observed results are due to these differences in experimental conditions.…”