How are DNA-unwinding enzymes regulated?

The fission-yeast helicase Pfh1 unwound DNA faster in the presence of spRim1, a mitochondrial single-stranded DNA-binding protein (SSB), but did not unwind a longer stretch in each run. Published in the Proceedings of the National Academy of Sciences (PNAS), the study found that Pfh1 repeatedly unwinds and rewinds short stretches of DNA, with contacts with both DNA strands and the use of adenosine triphosphate (ATP) regulating these processes.

Helicases are molecular motors that use ATP energy to separate the two strands of DNA. Pfh1, the only Pif1-family helicase in the fission yeast Schizosaccharomyces pombe, is essential for maintaining nuclear and mitochondrial genomes. Previous studies had also observed repeated unwinding and rewinding, but had not fully explained why unwinding stops after a short stretch, how rewinding occurs, or how accessory proteins regulate these processes.

Faster unwinding, unchanged run length

The researchers performed in vitro single-molecule experiments using purified proteins and nucleic-acid substrates. They tracked unwinding and rewinding by measuring changes in DNA hairpin length with optical tweezers, and also observed Pfh1 movement on single-stranded DNA using a combination of optical tweezers and confocal fluorescence microscopy. Under representative conditions, Pfh1 and spRim1 were each present at 2 nM. The team compared activity while varying ATP concentration and the force applied to the DNA.

With Pfh1 alone, an average of 22 ± 0.5 base pairs were unwound before rewinding occurred. Adding spRim1 did not significantly change processivity—the length unwound in a single run—which was 21 ± 0.5 base pairs. By contrast, the maximum forward unwinding rate, obtained by analyzing forward motion after excluding transient pauses and short backward movements, rose from 75 ± 5 to 115 ± 2 base pairs per second, an increase of about 1.5-fold. Thus, Pfh1 moved faster but did not unwind a longer stretch continuously.

spRim1 also increased the rewinding rate and shortened the wait until the next unwinding event. The researchers interpreted this as spRim1 binding to the DNA strand displaced during unwinding and competing with Pfh1 for contact with that strand. Human mitochondrial SSB similarly increased the unwinding rate of fission-yeast Pfh1. This finding supports regulation through SSB binding to DNA rather than through direct, species-specific binding between the proteins.

ATP-regulated rewinding and a backward-slippage model

The rewinding rate varied with ATP concentration and slowed substantially when a slowly hydrolyzed ATP analog was added. This shows that rewinding cannot be explained solely by DNA closing spontaneously after the protein dissociates; it also involves ATP use and regulation of Pfh1–DNA contacts.

The rewinding rate on a hybrid substrate in which part of the displaced strand was replaced with RNA was similar to that on a DNA substrate. Given previous findings that Pfh1 cannot move directionally along single-stranded RNA, this result is inconsistent with an explanation in which it switches to the opposite strand and translocates along it. The researchers proposed that Pfh1 temporarily loosens its grip on the strand along which it translocates, allowing the two strands to reanneal as the enzyme slips backward. They did not, however, rule out strand switching on other substrates.

These findings support a mechanochemical model in which contact with both strands helps unwinding while also limiting how far the enzyme proceeds. Whether the same regulation operates in cellular replication machinery, and how far it applies to human PIF1, remain questions for future study. The methods and results are available in the full text of the PNAS article.