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Here, we use ChAP-MS (chromatin affinity purification with mass spectrometry), for the affinity purification of a sequence-specific single-copy endogenous chromosomal locus containing a DNA double-strand break (DSB). We found multiple new histone post-translational modifications enriched on chromatin bearing a DSB from budding yeast. One of these, methylation of histone H3 on lysine 125, has not previously been reported. Among over 100 novel proteins enriched at a DSB were the phosphatase Sit4, the RNA pol II degradation factor Def1, the mRNA export protein Yra1 and the HECT E3 ligase Tom1. Each of these proteins was required for resistance to radiomimetics, and many were required for resistance to heat, which we show here to cause a defect in DSB repair in yeast. Yra1 and Def1 were required for DSB repair per se, while Sit4 was required for rapid inactivation of the DNA damage checkpoint after DSB repair. Thus, our unbiased proteomics approach has led to the unexpected discovery of novel roles for these and other proteins in the DNA damage response.
Pubmed ID: 29036368
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Commercial instrument and chemical vendor. Developer and manufacturer of specialized technological products for life science research and clinical diagnostics markets.
View all literature mentionsCentral repository for collection of functional information on proteins, with accurate and consistent annotation. In addition to capturing core data mandatory for each UniProtKB entry (mainly, the amino acid sequence, protein name or description, taxonomic data and citation information), as much annotation information as possible is added. This includes widely accepted biological ontologies, classifications and cross-references, and experimental and computational data. The UniProt Knowledgebase consists of two sections, UniProtKB/Swiss-Prot and UniProtKB/TrEMBL. UniProtKB/Swiss-Prot (reviewed) is a high quality manually annotated and non-redundant protein sequence database which brings together experimental results, computed features, and scientific conclusions. UniProtKB/TrEMBL (unreviewed) contains protein sequences associated with computationally generated annotation and large-scale functional characterization that await full manual annotation. Users may browse by taxonomy, keyword, gene ontology, enzyme class or pathway.
View all literature mentionsA quantitative proteomics software package for analyzing large-scale mass-spectrometric data sets. It is a set of algorithms that include peak detection and scoring of peptides, mass calibration, database searches for protein identification, protein quantification, and provides summary statistics.
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