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

Abstract

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.

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

“…Since this replication defect was independent of the ATPase/helicase activity located in the ordered C-terminal domain of Rrm3 (residues 250–723), we explored a possible involvement of the 230-residue, disordered N-terminal tail (Fig 3A, S4A Fig). The only motifs previously identified in this tail are a putative PCNA-interacting peptide (PIP) box between residues 35–42 [18] and a cluster of phosphorylated residues between S85 and S92 [17]. Deletion or mutation of the PIP-box ( rrm3-ΔN54 , rrm3-FFAA ) had no effect on DNA replication in HU, whereas deletion of the entire N-terminal tail ( rrm3-ΔN230 ) caused the same replication defect as deleting RRM3 ( rrm3Δ ) (Fig 3C).…”

“…A putative PCNA-interaction motif (PIP) between residues 35–42 [18] and a cluster of phosphorylated residues (P) between residues 85–95 [17] are indicated by gray boxes in the disordered tail. N-terminal tail truncations ( rrm3ΔN54 , rrm3ΔN142 , rrm3ΔN186 , rrm3ΔN212 , rrm3ΔN230 ) and point mutations designed to inactivate the PIP box ( rrm3-FFAA) and the Walker A motif of the helicase domain ( rrm3-K260A , rrm3-K260D ) were constructed.…”

“…Rrm3 possesses an N-terminal PCNA-interacting peptide (PIP) box, associates with the replication fork in vivo and is hyperphosphorylated by Rad53 under replication stress [1,17,18]. The replication damage that arises in the absence of Rrm3 causes constitutive, Mec3/Mec1/Rad9-dependent activation of the checkpoint kinase Rad53 [5,17,19,20].…”

“…The replication damage that arises in the absence of Rrm3 causes constitutive, Mec3/Mec1/Rad9-dependent activation of the checkpoint kinase Rad53 [5,17,19,20]. As a result, Dun1 kinase is activated, leading to degradation of the ribonucleotide reductase (RNR) inhibitor Sml1 and an increase in the dNTP pool [21,22].…”

Structure and function of Pif1 helicase

“…The Blackburn lab has shown that DSBs, but not stalled replication forks, result in Rad53 mediated phosphorylation of the C-terminus of ScPif1 at T763 and S766 which inhibits telomerase specifically at DSBs [31]. Both ScPif1 and Rrm3 are also phosphorylated in response to replication fork stalling in the N-terminal domain by Rad53 [103]. These phosphorylations inhibit ScPif1 and Rrm3 which prevents fork reversal and genome instability.…”