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

Abstract

In response to replication stress, a phospho-signaling cascade is activated and required for coordination of DNA repair and replication of damaged templates (intra-S-phase checkpoint) . How phospho-signaling coordinates the DNA replication stress response is largely unknown. We employed state-of-the-art liquid chromatography tandem-mass spectrometry (LC-MS/MS) approaches to generate high-coverage and quantitative proteomic and phospho-proteomic profiles during replication stress in yeast, induced by continuous exposure to the DNA alkylating agent methyl methanesulfonate (MMS) . We identified 32,057 unique peptides representing the products of 4296 genes and 22,061 unique phosphopeptides representing the products of 3183 genes. A total of 542 phosphopeptides (mapping to 339 genes) demonstrated an abundance change of greater than or equal to twofold in response to MMS. The screen enabled detection of nearly all of the proteins known to be involved in the DNA damage response, as well as many novel MMS-induced phosphorylations. We assessed the functional importance of a subset of key phosphosites by engineering a panel of phosphosite mutants in which an amino acid substitution prevents phosphorylation. In total, we successfully mutated 15 MMSresponsive phosphorylation sites in seven representative genes including APN1 (base excision repair); CTF4 and TOF1 (checkpoint and sister-chromatid cohesion); MPH1 (resolution of homologous recombination intermediates); RAD50 and XRS2 (MRX complex); and RAD18 (PRR). All of these phosphorylation site mutants exhibited MMS sensitivity, indicating an important role in protecting cells from DNA damage. In particular, we identified MMS-induced phosphorylation sites on Xrs2 that are required for MMS resistance in the absence of the MRX activator, Sae2, and that affect telomere maintenance.KEYWORDS mass spectrometry; phosphorylation; methyl methanesulfonate; DNA damage checkpoint; genetic interaction; homologous recombination; telomere; DNA damage response C ELLS 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 hallmarked 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 ; 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-media...

Meiotic DNA breaks activate a streamlined phospho-signaling response that largely avoids protein-level changes

“…This group contained many genes of the canonical gene expression response to DNA damage, including RNR2 , RNR3 , and RNR4 ( Elledge & Davis, 1987 , 1990 ; Huang & Elledge, 1997 ) ( Fig 5B ). This result was unexpected because the DNA damage–dependent transcriptional induction in mitotic cells directly results in a clear increase in protein levels ( Yao et al, 2003 ; Huang et al, 2016 ).…”

Discovery of Arginine Methylation, Phosphorylation, and Their Co-occurrence in Condensate-Associated Proteins in Saccharomyces cerevisiae

“…The following eight yeast phosphoproteomic datasets were searched for the presence of phospho-methylarginine peptides: PXD001368, PXD002344, PXD013453, PXD013782, PXD015235, PXD015575, PXD016519, and Swaney et al Data were searched in Proteome Discoverer (v.2.3) using Mascot, as above, with the mass tolerances and modifications listed in Table S1.…”