Abstract
We have used a novel method to activate the DNA damage S-phase checkpoint response in Saccharomyces cerevisiae to slow lagging-strand DNA replication by exposing cells expressing a drug-sensitive DNA polymerase δ (L612M-DNA pol δ) to the inhibitory drug phosphonoacetic acid (PAA). PAA-treated pol3-L612M cells arrest as large-budded cells with a single nucleus in the bud neck. This arrest requires all of the components of the S-phase DNA damage checkpoint: Mec1, Rad9, the DNA damage clamp Ddc1-Rad17-Mec3, and the Rad24-dependent clamp loader, but does not depend on Mrc1, which acts as the signaling adapter for the replication checkpoint. In addition to the above components, a fully functional mismatch repair system, including Exo1, is required to activate the S-phase damage checkpoint and for cells to survive drug exposure. We propose that mismatch repair activity produces persisting single-stranded DNA gaps in PAA-treated pol3-L612M cells that are required to increase DNA damage above the threshold needed for checkpoint activation. Our studies have important implications for understanding how cells avoid inappropriate checkpoint activation because of normal discontinuities in lagging-strand replication and identify a role for mismatch repair in checkpoint activation that is needed to maintain genome integrity.
Highlights
EUKARYOTIC cells have the ability to detect DNA damage and stalled replication forks, and, if problems are found, checkpoints are triggered to control cell cycle progression, induce DNA repair enzymes, stabilize replication forks, remodel chromatin, modulate gene expression, and many other activities
About 70% of the nuclear genome of phosphonoacetic acid (PAA)-arrested pol3-L612M cells was replicated as determined by comparing the amount of [3H]dTMP-labeled DNA per cell for cells not exposed to PAA to the amount of label per cell in cells exposed to PAA
Survival requires all components of the DNA damage checkpoint, including Mec1, the signaling adapter Rad9, and the PCNAlike DNA damage clamp (Ddc1-Rad17-Mec3) and damage clamp loader (Rad24) complex (Figure 3; Figure S2 and Figure S3). pol3-L612M cells deficient in any of these proteins are highly sensitive to PAA and, as shown for rad24 pol3-L612M cells, fail to activate the DNA damage checkpoint as demonstrated by the failure of cells to arrest cell cycle progression, to curb DNA replication and to phosphorylate Rad53 and by subsequent loss of cell viability (Figures 2–4). pol3-L612M cells have less dependence on Mrc1, the checkpoint adapter for stalled replication forks (Figure 3)
Summary
EUKARYOTIC cells have the ability to detect DNA damage and stalled replication forks, and, if problems are found, checkpoints are triggered to control cell cycle progression, induce DNA repair enzymes, stabilize replication forks, remodel chromatin, modulate gene expression, and many other activities. For the “processing” model, MMR DNA degradation activity is proposed to generate persisting ssDNA gaps that produce DSBs when encountered by advancing replication forks (Mojas et al 2007) These proposed roles of MMR in checkpoint activation, are paradoxical since checkpoint activation is a mechanism generally thought to help cells survive DNA damage, but the absence of MMR activity often renders mammalian cells less sensitive to killing by several DNA-damaging agents (reviewed by Jiricny 2006; Hsieh and Yamane 2008).
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