Dataset Information

Ndd1 phosphorylation pattern upon DNA damage

ABSTRACT: The transcriptional activator NDD1 was purified from wt and rad53 or mec1 mutant treated with hydroxyurea or methyl-methanesulfonate via HTB-tag , digested directly from the streptavidin beads, and then subjected to LC-MSMS analysis. Beads were alternatively digested with trypsin and chymotrypsin for better sequence coverage.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Dorothea Anrather

PROVIDER: PXD000405 | Pride | 2013-12-19

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	120921V_Ndd1_mec1_HU_chymo.msf	Msf
	120921V_Ndd1_mec1_HU_chymo.pep.xml	Pepxml
	120921V_Ndd1_mec1_HU_chymo.raw	Raw
	120921V_Ndd1_mec1_HU_tryp.msf	Msf
	120921V_Ndd1_mec1_HU_tryp.pep.xml	Pepxml

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Publications

Genotoxic stress prevents Ndd1-dependent transcriptional activation of G2/M-specific genes in Saccharomyces cerevisiae.

Yelamanchi Syam Kumar SK Veis Jiri J Anrather Dorothea D Klug Helene H Ammerer Gustav G

Molecular and cellular biology 20131209 4

Downregulation of specific transcripts is one of the mechanisms utilized by eukaryotic checkpoint systems to prevent cell cycle progression. Here we identified and explored such a mechanism in the yeast Saccharomyces cerevisiae. It involves the Mec1-Rad53 kinase cascade, which attenuates G(2)/M-specific gene transcription upon genotoxic stress. This inhibition is achieved via multiple Rad53-dependent inhibitory phosphorylations on the transcriptional activator Ndd1 that prevent its chromatin rec ...[more]

PMID: 24324010

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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.