Checkpoint phosphorylation sites on budding yeast Rif1 protect nascent DNA from degradation by Sgs1-Dna2

Abstract

12In budding yeast the Rif1 protein is important for protecting nascent DNA at blocked 13 replication forks, but the mechanism has been unclear. Here we show that budding yeast 14 Rif1 must interact with Protein Phosphatase 1 to protect nascent DNA. In the absence of 15 Rif1, removal of either Dna2 or Sgs1 prevents nascent DNA degradation, implying that Rif1 16 protects nascent DNA by targeting Protein Phosphatase 1 to oppose degradation by the 17 Sgs1-Dna2 nuclease-helicase complex. This functional role for Rif1 is conserved from yeast 18 to human cells. Yeast Rif1 was previously identified as a target of phosphorylation by the 19 Tel1/Mec1 checkpoint kinases, but the importance of this phosphorylation has been 20 unclear. We find that nascent DNA protection depends on a cluster of Tel1/Mec1 consensus 21 phosphorylation sites in the Rif1 protein sequence, indicating that the intra-S phase 22Author summary 27 Genome instability is a leading factor contributing to cancer. Maintaining efficient error-28 free replication of the genome is key to preventing genome instability. During DNA 29 replication, replication forks can be stalled by external and intrinsic obstacles, leading to 30 processing of nascent DNA ends to enable replication restart. However, the nascent DNA 31 must be protected from excessive processing to prevent terminal fork arrest, which could 32 potentially lead to more serious consequences including failure to replicate some genome 33 sequences. Using a nascent DNA protection assay we have investigated the role of the 34 budding yeast Rif1 protein at blocked replication forks. We find that Rif1 protects nascent 35DNA through a mechanism that appears conserved from yeast to humans. We show that 36 budding yeast Rif1 protects nascent DNA by targeting Protein Phosphatase 1 activity to 37 prevent degradation of nascent DNA by the Sgs1-Dna2 helicase-nuclease complex. 38Furthermore, we find that Rif1 phosphorylation by the checkpoint pathway during 39 replication stress is crucial for this function. Our results indicate that the S phase checkpoint 40 machinery acts by phosphorylating Rif1 to protect nascent DNA, providing important clues 41 concerning the conserved role of Rif1 in regulating events when replication is challenged. 42 budding yeast Saccharomyces cerevisiae [1]. While its telomere length regulation function 52 appears to be specific to yeast [2], other roles of Rif1 are conserved in eukaryotes [3]. One 53 apparently conserved function of Rif1 is promotion of double-stranded break (DSB) repair 54 through non-homologous end joining (NHEJ). Rif1 drives DSB repair toward NHEJ by 55 protecting 5' ends from resection that would favour homology-directed repair (HDR), in a 56 function that appears to be conserved from budding yeast to human cells [4][5][6][7]. 57Mammalian Rif1 also plays a role in programmed genomic rearrangements in mammalian 58 cells, such as immunoglobulin class switching, which is a specialised form of NHEJ [4,5]. 59 Another conserved function of Rif1 is control of the initiati...

Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

“…In this regard, the IDR of Saccharomyces cerevisiae Rif1 contains a cluster of seven SQ/TQ consensus motifs for the ATM/ATR yeast homologs Tel1/Mec1, some of which have been reported to be phosphorylated in vivo ( Smolka et al, 2007 ; Sridhar et al, 2014 ; Wang et al, 2018 ). Interestingly, a recent bioRxiv manuscript showed that abrogation of phosphorylation at these seven SQ/TQ sites in yeast Rif1 impaired DNA replication fork protection after treatment with HU ( Monerawela et al, 2020 ). Although RIF1 IDRs exhibit low conservation across evolution, the identification of a cluster of SQ/TQ motifs whose phosphorylation influences fork protection in both mammalian and yeast RIF1 hints at an evolutionary conserved mechanism, and underlying molecular feature, for the regulation of nascent DNA processing under conditions of DNA replication stress.…”

Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

“…In this regard, the IDR of S. cerevisiae Rif1 contains a cluster of seven SQ/TQ consensus motifs for the ATM/ATR yeast homologs Tel1/Mec1, some of which have been reported to be phosphorylated in vivo (Smolka et al, 2007;Sridhar et al, 2014;Wang et al, 2018). Interestingly, a recent bioRxiv manuscript showed that abrogation of phosphorylation at these seven SQ/TQ sites in yeast Rif1 impaired DNA replication fork protection after treatment with HU (Monerawela et al, 2020). Although RIF1 IDRs exhibit low conservation across evolution, the identification of a cluster of SQ/TQ motifs whose phosphorylation influences fork protection in both mammalian and yeast RIF1 hints at an evolutionary conserved mechanism, and underlying molecular feature, for the regulation of nascent DNA processing under conditions of DNA replication stress.…”