Academic journal article Genetics

DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces Cerevisiae

Academic journal article Genetics

DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces Cerevisiae

Article excerpt

CELLS utilize excision repair and DNA damage tolerance pathways without significant delay of the cell cycle to address low levels of DNA base damage (Hishida et al. 2009; Huang et al. 2013), while more extensive damage is hall-marked by the activation of additional checkpoints, prolonged cell cycle arrest, and utilization of additional repair mechanisms (Lazzaro et al. 2009). A classic example of an agent that elicits a profoundly different DNA damage response (DDR) at high and low doses is the monofunctional alkylating agent methyl methanesulfonate (MMS) (Friedberg and Friedberg 2006; Hanawalt 2015). At low doses, the MMS lesions are well tolerated by wild-type cells and do not elicit any discernible sensitivity (Huang et al. 2013); however, at higher concentrations, MMS-induced DNA damage present during the S phase leads to prolonged replication fork stall, a phenomenon termed "replication stress" (Shimada et al. 2002; Zeman and Cimprich 2013). As a result of replication stress, cells synchronize into a lengthened S phase due to a kinase-mediated checkpoint response (Paulovich and Hartwell 1995; Murakami-Sekimata et al. 2010).

Much of the known signaling in the DDR is mediated by a group of highly conserved checkpoint kinases (e.g., ATR/ Mec1, ATM/Tel1, Chk2/Rad53, Chk1), which activate an extensive phospho-signaling network to enhance DNA repair capacity as well as induce cell cycle delay at G1, intra-S, or G2/M to allow additional time for cells to deal with higher doses of DNA damage (Weinert and Hartwell 1988; Siede et al. 1993; Paulovich and Hartwell 1995). In Saccharomyces cerevisiae, the intra-S-phase checkpoint is mediated by the serine/threonine protein kinases Mec1 and Tel1 (Paulovich and Hartwell 1995; Zeman and Cimprich 2013). Mec1 plays the predominant role in the activation of the intra-S-phase checkpoint, whereas Tel1 plays a backup role (Weinert et al. 1994; Greenwell et al. 1995). The long stretches of single-stranded DNA (ssDNA) exposed during replication fork stalling after DNA damage contribute to the activation of Mec1 and induction of the intra-S-phase checkpoint (Tercero et al. 2003; MacDougall et al. 2007).

Activation of the Mec1 kinase leads to activation of two well-known, bifurcated pathways: the Rad9-mediated DNA-damage checkpoint (DDC) pathway, and the Mrc1/Tof1/Ctf4/Csm3-mediated S phase-specific DNA-replication checkpoint (DRC) (Alcasabas et al. 2001; Katou et al. 2003; Uzunova et al. 2014). Similar to the Mec1/Tel1 relationship, the Rad9-mediated DDC pathway is required for MMS resistance, whereas the Mrc1-mediated DRC plays a backup role in MMS resistance (Foss 2001). Phosphorylations of Rad9 and Mrc1 in turn facilitate phosphorylation of the downstream checkpoint kinases (Rad53 and Chk1)(Vialardet al. 1998; Sanchez et al. 1999; Alcasabas et al. 2001), which, in turn, phosphorylate additional substrates, including Pds1 and Cdc5 polo-kinase, both of which contribute to cell cycle delay (Sanchez et al. 1999). Rad53 also phosphorylates and activates another kinase, Dun1, which contributes to the hyper-phosphorylation and inactivation of the transcriptional repressor Crt1 and leads to increased expression of genes related to DNA repair, including RNR (ribonucleotide-diphosphate reductase) genes (Huang et al. 1998).

Many downstream DNA repair proteins are reported to be phosphorylated during checkpoint activation, including proteins involved in post-replication repair (PRR), homologous recombination (HR), DNA replication, DNA repair, histone modification, and chromatin remodeling (Smolka et al. 2007; Chen et al. 2010; Bastos de Oliveira et al. 2015). For example, phosphorylation of Rev1 by Mec1 increases the proficiency of Polz-mediated translesion synthesis (Pages et al. 2009), which together with the template-switch subpathways of PRR are important in dealing with replication stress, because the lesion-containing ssDNA resulting from a replication fork stall would not be subject to excision repair (Yang et al. …

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