Unwinding G4s: Effects of G-Quadruplex DNA and Pif1 Helicases on Replication Fork Progression in S. cerevisiae

Abstract

Replication fork progression is essential to cell division. Barriers to fork progression include G-quadruplexes (G4s), DNA structures with four triplets of Hoogsteen bonded guanines. During cell division, helicases, such as those in the Pif1 family, help remove or resolve barriers, such as G4s, to promote fork progression. Pif1 helicases have been shown to resolve G4s in vitro. This paper seeks to provide further evidence for the formation of G4s and the effects of Pif1 helicases on G4s in vivo. G4s’ effects on double-strand break (DSB) frequency were tested in pif1m-2 rrm3∆ S. cerevisiae, mutant for both its Pif1 helicases. ChIP-identified Pif1-binding G4 sequences were inserted within direct repeat constructs into the ADE2 locus of pif1m-2 rrm3∆ yeast. By direct repeat assays, recombination frequencies were compared between yeast with direct repeat inserts with and without G4 sequences. Compared to the no-G4 insert strains, several G4 insert strains showed differential frequency of recombination, an indicator of DSBs. Counterintuitively, the G4s seem to decrease DSB frequency and in an increasing manner with increasing G4 stability. This trend suggests the possible presence of an alternative mechanism for G4s’ effects on fork stalling in pif1m-2 rrm3∆ mutants.