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Mec1 is activated at the onset of normal S phase by low dNTP pools impeding DNA replication [sequencing]

ABSTRACT: The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1–Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because dNTP levels present in G1 phase are not sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use dNTP shortage to detect the onset of DNA replication and activate the Mec1–Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE136602 | GEO | 2020/03/17

REPOSITORIES: GEO

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Project description:Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, ChIP on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double deletion of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD51 single deletion. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed. Keywords: ChIP-chip â¢ The goal of the experiment Genome-wide localization of Rad52 binding sites in Saccharomyces cerevisiae â¢ Experimental factor Distribution of Rad52 on chromosome III, IV, and V and the right arm of chromosome VI Strain: wild type, rad53 mutant, and rad53 siz2 mutant (W303 background, expressing myc tagged protein from its native promoter) Cell condition 1: G1 arrest with alpha-factor Cell condition 2: HU treatment â¢ Experimental design ChIP analyses: ChIP using anti-c-Myc antibody. ChIP-chip analyses: In all cases, hybridization data of ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F) were used. â¢ Quality control steps taken Confirmation of several loci by quantitative real time PCR. Wild type cells expressing non-tagged Rad52 were used as a negative control of DNA amplification.

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Mec1 is activated at the onset of normal S phase by low dNTP pools impeding DNA replication [sequencing]

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