Rad53 arrests leading and lagging strand DNA synthesis via distinct mechanisms in response to DNA replication stress

Abstract

DNA replication stress threatens ordinary DNA synthesis. The evolutionarily conserved DNA replication stress response pathway involves sensor kinase Mec1/ATR, adaptor protein Mrc1/Claspin, and effector kinase Rad53/Chk1, which spurs a host of changes to stabilize replication forks and maintain genome integrity. DNA replication forks consist of largely distinct sets of proteins at leading and lagging strands that function autonomously in DNA synthesis in vitro. In this article, we discuss eSPAN and BrdU‐IP‐ssSeq, strand‐specific sequencing technologies that permit analysis of protein localization and DNA synthesis at individual strands in budding yeast. Using these approaches, we show that under replication stress Rad53 stalls DNA synthesis on both leading and lagging strands. On lagging strands, it stimulates PCNA unloading, and on leading strands, it attenuates the replication function of Mrc1‐Tof1. We propose that in doing so, Rad53 couples leading and lagging strand DNA synthesis during replication stress, thereby preventing the emergence of harmful ssDNA.

Keeping the strands together: Rad53 regulation of fork symmetry promotes replication stability

“…These direct links to cell proliferation and mutational avoidance make DNA replication checkpoints important to genome stability. He and Zhang's Hypothesis explores how DNA replication complexes are maintained during replication instability and in an unstressed S‐phase 1 …”

“…Replication forks are the functional unit of DNA replication, with three main activities: synthesis by leading and lagging strand polymerases; unwinding double stranded DNA into single stranded template by DNA helicase components; and, stabilizing synthesis and unwinding through a fork protection complex. Functional differences between leading and lagging strands have been characterized using molecular and genomic methods (e.g., Refs 1–3 …”

“…He and Zhang describe how Rad53 impacts three distinct mechanisms that maintain fork symmetry and stability. Their work considers endogenous instability, and replication stress induced by hydroxyurea, using budding yeast cultures deficient in the DNA replication checkpoint (i.e., rad53) 1 . First, activated Rad53 decreases lagging strand processivity by removing proliferating cell nuclear antigen (PCNA).…”

“…He and Zhang note that orthologous Rad53 pathways are similar in metazoan models. However, dNTP regulation directly impacts replication stress 1 and is significantly different in Saccharomyces cerevisiae compared to mammals. Metazoan cells possess additional replication checkpoint regulatory factors.…”

“…Functional differences between leading and lagging strands have been characterized using molecular and genomic methods (e.g., Refs. [1][2][3] ). Decoupled leading and lagging strand synthesis is a potential symptom of replication distress.…”