Differential chromatin proteomics of the MMS-induced DNA damage response in yeast

被引：16
作者：
Kim D.R. [1 ]
Gidvani R.D. [1 ]
Ingalls B.P. [2 ]
Duncker B.P. [1 ]
McConkey B.J. [1 ]
机构：
[1] Department of Biology, University of Waterloo, Waterloo, ON
[2] Department of Applied Mathematics, University of Waterloo, Waterloo, ON
来源：
Proteome Science | / 9卷 / 1期
基金：
加拿大健康研究院;
关键词：
Chromatin; Differential; Dige; Dna damage; Fractionation; Mms; Proteomics;
D O I：
10.1186/1477-5956-9-62
中图分类号：
学科分类号：
摘要：
Background: Protein enrichment by sub-cellular fractionation was combined with differential-in-gel-electrophoresis (DIGE) to address the detection of the low abundance chromatin proteins in the budding yeast proteome. Comparisons of whole-cell extracts and chromatin fractions were used to provide a measure of the degree of chromatin association for individual proteins, which could be compared across sample treatments. The method was applied to analyze the effect of the DNA damaging agent methyl methanesulfonate (MMS) on levels of chromatin-associated proteins.Results: Up-regulation of several previously characterized DNA damage checkpoint-regulated proteins, such as Rnr4, Rpa1 and Rpa2, was observed. In addition, several novel DNA damage responsive proteins were identified and assessed for genotoxic sensitivity using either DAmP (decreased abundance by mRNA perturbation) or knockout strains, including Acf2, Arp3, Bmh1, Hsp31, Lsp1, Pst2, Rnr4, Rpa1, Rpa2, Ste4, Ycp4 and Yrb1. A strain in which the expression of the Ran-GTPase binding protein Yrb1 was reduced was found to be hypersensitive to genotoxic stress.Conclusion: The described method was effective at unveiling chromatin-associated proteins that are less likely to be detected in the absence of fractionation. Several novel proteins with altered chromatin abundance were identified including Yrb1, pointing to a role for this nuclear import associated protein in DNA damage response. © 2011 Kim et al; licensee BioMed Central Ltd.
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