Rad53-Mediated Regulation of Rrm3 and Pif1 DNA Helicases Contributes to Prevention of Aberrant Fork Transitions under Replication Stress

•Rrm3 and Pif1 promote fork reversal and ssDNA gaps at stalled forks in rad53 cells•Rrm3 and Pif1 associate with stalled DNA replication forks•Rad53 phosphorylates Rrm3 and Pif1 at stalled forks•Rrm3 and Pif1 promote chromosome fragility in hydroxyurea-treated rad53 cells

SummaryReplication stress activates the Mec1ATR and Rad53 kinases. Rad53 phosphorylates nuclear pores to counteract gene gating, thus preventing aberrant transitions at forks approaching transcribed genes. Here, we show that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites, are detrimental in rad53 mutants experiencing replication stress. Rrm3 and Pif1 ablations rescue cell lethality, chromosome fragmentation, replisome-fork dissociation, fork reversal, and processing in rad53 cells. Through phosphorylation, Rad53 regulates Rrm3 and Pif1; phospho-mimicking rrm3 mutants ameliorate rad53 phenotypes following replication stress without affecting replication across pausing elements under normal conditions. Hence, the Mec1-Rad53 axis protects fork stability by regulating nuclear pores and DNA helicases. We propose that following replication stress, forks stall in an asymmetric conformation by inhibiting Rrm3 and Pif1, thus impeding lagging strand extension and preventing fork reversal; conversely, under unperturbed conditions, the peculiar conformation of forks encountering pausing sites would depend on active Rrm3 and Pif1.

Graphical AbstractFigure optionsDownload full-size imageDownload as PowerPoint slide