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.

Keywords: ATR; DNA replication; DNA replication checkpoint; Mec1; Rad53; eSPAN; strand-specific sequencing.

Figure 1.

(a) A graphical model of Rad53-mediated fork stalling upon HU-induced replication stress. On lagging strands, PCNA is unloaded, which effectively halts the DNA synthesis by Pol δ. On leading strands, Rad53 phosphorylation of Mrc1 slows down leading strand DNA synthesis. (b) A schematic for Rad53 to regulate PCNA unloading via Elg1 phosphorylation.

Figure 2.

A schematic of the Mec1-Mrc1-Rad53 pathway to halt DNA synthesis under replication stress, highlighting the dual roles of Rad53 in PCNA unloading and in the attenuation of Mrc1-Tof1’s replication function.