Academic journal article Genetics

Drug-Sensitive DNA Polymerase [Delta] Reveals a Role for Mismatch Repair in Checkpoint Activation in Yeast

Academic journal article Genetics

Drug-Sensitive DNA Polymerase [Delta] Reveals a Role for Mismatch Repair in Checkpoint Activation in Yeast

Article excerpt

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 d (L612M-DNA pol d) 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.

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. Coordinated action of these processes prevents cell death while preserving genome integrity; however, if surveillance and repair activities are not successful, the resulting genomic instability produces cell death or is mutagenic and even potentially carcinogenic in human cells (Hartwell and Kastan 1994; Myung et al. 2001; Nyberg et al. 2002; Brown et al. 2003). Given the multitude of genotoxic lesions that can be produced by endogenous and exogenous agents and the formation of aberrant DNA structures that can occur during normal DNA replication (Tourrière and Pasero 2007), it is not surprising that S-phase checkpoint activation and responses are complex.

Briefly, checkpoint pathways are protein kinase cascades that begin with sensing DNA damage in the context of single-stranded DNA (ssDNA) that is coated with ssDNA binding protein (RPA) (Figure 1). The PCNA-like Ddc1- Rad17-Mec3 clamp complex (the Saccharomyces cerevisiae 9-1-1 DNA damage clamp) is loaded near DNA damage by the Rad24-clamp loader complex. Mec1/Ddc2 (yeast ATR/ ATRIP) is loaded onto ssDNA regions at gaps and the resected ssDNA ends produced at double-strand breaks (DSBs) (Zou and Elledge 2003). Mec1 phosphorylates Ddc2, and colocalization with the DNA damage clamp allows phosphorylation of Ddc1. Mec1 also phosphorylates Rad9, which is a signaling adapter that brings Mec1 and Rad53 together and facilitates phosphorylation of Rad53, the yeast ortholog of the effector kinase Chk2 (Sweeney et al. 2005). Rad9 also facilitates the phosphorylation of Chk1. Mrc1 (yeast claspin) appears to fulfill a similar role as Rad9 in Rad53 phosphorylation, but in response to stalled replication forks (Alcasabus et al. 2001). Thus, the DNA damage checkpoint is mediated by Rad9 and the replication checkpoint by Mrc1.

Rad53 phosphorylation (Figure 1) is a key step in the signal transduction cascade (Tercero and Diffley 2001; Nyberg et al. 2002). Once activated, Rad53 phosphorylates protein targets including Dun1, which in turn phosphorylates proteins that lead to the up-regulation of deoxynucleoside triphosphate (dNTP) synthesis and the transcription of DNA repair genes (Huang et al. 1998; Bashkirov et al. 2000; Zhao and Rothstein 2002; Ahnesorg and Jackson 2007). Rad53 also acts either alone or in combination with Chk1 to arrest cells at G2/M, which prevents the premature segregation of damaged or broken chromosomes (reviewed in Hsieh and Yamane 2008; Branzei and Foiani 2009; Schleker et al. …

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