RPA and Pif1 cooperate to remove G-rich structures at both leading and lagging strand.
In  Saccharomyces cerevisiae , the absence of Pif1 helicase induces the instability of G4-containing CEB1 minisatellite during leading strand but not lagging strand replication. We report that RPA and Pif1 cooperate to maintain CEB1 stability when the G4 forming strand is either on the leading or lagging strand templates. At the leading strand, RPA acts in the same pathway as Pif1 to maintain CEB1 stability. Consistent with this result, RPA co-precipitates with Pif1. This association between Pif1 and RPA is affected by the  rfa1-D228Y  mutation that lowers the affinity of RPA in particular for G-rich single-stranded DNA. At the lagging strand, in contrast to  pif1 Δ, the  rfa1-D228Y  mutation strongly increases the frequency of CEB1 rearrangements. We explain that Pif1 is dispensable at the lagging strand DNA by the ability of RPA by itself to prevent formation of stable G-rich secondary structures during lagging strand synthesis. Remarkably, overexpression of Pif1 rescues the instability of CEB1 at the lagging strand in the  rfa1-D228Y  mutant indicating that Pif1 can also act at the lagging strand. We show that the effects of the  rfa1-D228Y  ( rpa1-D223Y  in fission yeast) are conserved in  Schizosaccharomyces pombe . Finally, we report that RNase H1 interacts in a DNA-dependent manner with RPA in budding yeast, however overexpression of RNase H1 does not rescue CEB1 instability observed in  pif1 Δ and  rfa1-D228Y  mutants. Collectively these results add new insights about the general role of RPA in preventing formation of DNA secondary structures and in coordinating the action of factors aimed at resolving them.
Maestroni L, Audry J, Luciano P, Coulon S, Géli V, Corda Y