All eukaryotes contain the accessory helicase Pif1 which plays a role in genome maintenance processes. For example, Pif1 is involved in DNA replication across G-quadruplexes and break-induced replication repair. Inactivation of Pif1 causes mitochondrial myopathy in mice with respiratory chain deficiency as a result of genome instability. Genome instability is often linked to the inability of the DNA replication complex (replisome) to bypass obstacles. We reveal a previously unknown role for Pif1 in replication barrier bypass[1].
We use an inactive Cas9 (dCas9) as a barrier to replication[2]. The dCas9 forms a protein–R-loop construct similar to stalled RNA polymerases. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 works in concert with the replisome, to enable replication bypass of the barrier. We developed a novel single-molecule fluorescence visualisation technique[3] that allows us to study the fate of the dCas9 block. Our results show that Pif1 facilitates replication bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop.
These results suggest that Pif1 can enable the removal of stalled RNA polymerases and thus could play an important role during replication–transcription conflicts. We propose that Pif1, together with the replisome, can support the displacement of both R-loops and protein blocks. Therefore, we anticipate that this activity could also play a role in the removal of other protein roadblocks, outside the context of R-loops.