Inaccurate repair of DNA double-strand breaks (DSBs) can lead to DNA mutation and chromosome rearrangements, causing human diseases such as cancer. Although we know the basic mechanisms of DSB repair, the added complexities in the chromatin context are unclear. This is partially due to the lack of unbiased systems for identifying proteins and post-translational modifications (PTMs) involved in DSB repair. In this work, we established a novel method, termed DSB-ChAP-MS (Double Strand Break-Chromatin Affinity Purification with Mass Spectrometry), for the affinity purification of a sequence-specific single copy endogenous chromosomal locus containing a DSB, followed by the proteomic identification of enriched proteins and histone PTMs. Providing validation of the DSB-ChAP-MS approach, we found many histone PTMs that had been previously implicated in the DNA damage response, as well as multiple new histone PTMs 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 the 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. 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.
","abstract_html":"<p>Inaccurate repair of DNA double-strand breaks (DSBs) can lead to DNA mutation and chromosome rearrangements, causing human diseases such as cancer. Although we know the basic mechanisms of DSB repair, the added complexities in the chromatin context are unclear. This is partially due to the lack of unbiased systems for identifying proteins and post-translational modifications (PTMs) involved in DSB repair. In this work, we established a novel method, termed DSB-ChAP-MS (Double Strand Break-Chromatin Affinity Purification with Mass Spectrometry), for the affinity purification of a sequence-specific single copy endogenous chromosomal locus containing a DSB, followed by the proteomic identification of enriched proteins and histone PTMs. Providing validation of the DSB-ChAP-MS approach, we found many histone PTMs that had been previously implicated in the DNA damage response, as well as multiple new histone PTMs 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 the 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. 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.</p>","abstract_has_math":false,"creators":["Wang, Pingping","0000-0001-6203-5725
"],"institution":null,"degree_name":"Doctor of Philosophy (PhD)","degree_level":"Dissertation (PhD)","degree_discipline":null,"degree_department":null,"school":null,"contributors":["Jessica Tyler","Bin Wang","Xiaobing Shi"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2017,"date_issued":"2017-05-01T07:00:00Z","date_published":"2017-05-01T07:00:00Z","updated_at":"2026-07-24T05:49:54Z","subjects":["DSB","PTM","Affinity Purification","Mass Spectrometry","Yeast","DNA repair","Checkpoint","Biology","Cell Biology","Life Sciences","Medicine and Health Sciences","Microbiology","Molecular Genetics"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.library.tmc.edu/utgsbs_dissertations/731","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Jessica Tyler","Bin Wang","Xiaobing Shi"]},{"key":"dc:creator","label":"Author","values":["Wang, Pingping","0000-0001-6203-5725
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"]},{"key":"dc:title","label":"Title","values":["Proteomic Identification of Histone Post-Translational Modifications Induced By Dna Double-Strand Breaks and Novel Proteins Involved In The Dna Damage Response"]}]}],"canonical_facts":{"dc:contributor":["Jessica Tyler","Bin Wang","Xiaobing Shi"],"dc:creator":["Wang, Pingping","0000-0001-6203-5725
"],"dc:date.available":["2018-03-07T08:00:00Z"],"dc:description.abstract":["Inaccurate repair of DNA double-strand breaks (DSBs) can lead to DNA mutation and chromosome rearrangements, causing human diseases such as cancer. Although we know the basic mechanisms of DSB repair, the added complexities in the chromatin context are unclear. This is partially due to the lack of unbiased systems for identifying proteins and post-translational modifications (PTMs) involved in DSB repair. In this work, we established a novel method, termed DSB-ChAP-MS (Double Strand Break-Chromatin Affinity Purification with Mass Spectrometry), for the affinity purification of a sequence-specific single copy endogenous chromosomal locus containing a DSB, followed by the proteomic identification of enriched proteins and histone PTMs. Providing validation of the DSB-ChAP-MS approach, we found many histone PTMs that had been previously implicated in the DNA damage response, as well as multiple new histone PTMs 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 the 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. 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.
"],"dc:identifier":["https://digitalcommons.library.tmc.edu/utgsbs_dissertations/731"],"dc:subject":["DSB","PTM","Affinity Purification","Mass Spectrometry","Yeast","DNA repair","Checkpoint","Biology","Cell Biology","Life Sciences","Medicine and Health Sciences","Microbiology","Molecular Genetics"],"dc:title":["Proteomic Identification of Histone Post-Translational Modifications Induced By Dna Double-Strand Breaks and Novel Proteins Involved In The Dna Damage Response"],"thesis:degree_level":["Dissertation (PhD)"],"thesis:degree_name":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-24T05:49:54Z"}