Replication fork slowing and stalling are distinct, checkpoint-independent consequences of replicating damaged DNA

Abstract

In response to DNA damage during S phase, cells slow DNA replication. This slowing is orchestrated by the intra-S checkpoint and involves inhibition of origin firing and reduction of replication fork speed. Slowing of replication allows for tolerance of DNA damage and suppresses genomic instability. Although the mechanisms of origin inhibition by the intra-S checkpoint are understood, major questions remain about how the checkpoint regulates replication forks: Does the checkpoint regulate the rate of fork progression? Does the checkpoint affect all forks, or only those encountering damage? Does the checkpoint facilitate the replication of polymerase-blocking lesions? To address these questions, we have analyzed the checkpoint in the fission yeast Schizosaccharomyces pombe using a single-molecule DNA combing assay, which allows us to unambiguously separate the contribution of origin and fork regulation towards replication slowing, and allows us to investigate the behavior of individual forks. Moreover, we have interrogated the role of forks interacting with individual sites of damage by using three damaging agents—MMS, 4NQO and bleomycin—that cause similar levels of replication slowing with very different frequency of DNA lesions. We find that the checkpoint slows replication by inhibiting origin firing, but not by decreasing fork rates. However, the checkpoint appears to facilitate replication of damaged templates, allowing forks to more quickly pass lesions. Finally, using a novel analytic approach, we rigorously identify fork stalling events in our combing data and show that they play a previously unappreciated role in shaping replication kinetics in response to DNA damage.

Mapping DNA damage‐dependent genetic interactions in yeast via party mating and barcode fusion genetics

“…MMS and 4NQO are members of different drug classes, but both are DNA alkylating agents (Xiao & Chow, ; Svensson et al , ). Both MMS and 4NQO cause checkpoint‐modulated fork stalling (Minca & Kowalski, ; Iyer & Rhind, ) that appears to facilitate replication of damaged templates allowing forks to quickly pass lesions (Iyer & Rhind, ). Furthermore, strains carrying deletion of genes involved in postreplication repair (PRR) processes, such as MMS2, RAD5, and UBC13, are significantly hypersensitive to both MMS and 4NQO (Lee et al , ), suggesting that PRR acts on both MMS and 4NQO lesions.…”

A tel2 Mutation That Destabilizes the Tel2-Tti1-Tti2 Complex Eliminates Rad3ATR Kinase Signaling in the DNA Replication Checkpoint and Leads to Telomere Shortening in Fission Yeast

“…DNA replication can also be perturbed by DNA damage such as those caused by methyl methanesulfonate (MMS) or UV light. Unlike HU, which slows forks globally, DNA damage pauses a subset of on-going forks at the damage sites on the leading strand template (20). In addition, DNA damage, if occurs outside S phase, provokes the DNA damage checkpoint (DDC) responses ( Fig.…”

The Human RAD5 Homologs, HLTF and SHPRH, Have Separate Functions in DNA Damage Tolerance Dependent on the DNA Lesion Type

“…However, CHK2, as well as other factors, might also be involved in regulation of origin firing. For example, in a recent study in yeast, inhibition of origin firing was observed in response to MMS before CHK1 was phosphorylated [32]. Interestingly, a similar phenotype, i.e., maintained replication upon MMS treatment, is reported in yeast cells with reduced Rad53-levels, the functional CHK2 homolog in yeast [33].…”