Characterization o f the DNA Ligase IV and XRCC4 complex in the DNA double-strand break repair

Hdl Handle:
http://hdl.handle.net/10675.2/347146
Title:
Characterization o f the DNA Ligase IV and XRCC4 complex in the DNA double-strand break repair
Authors:
Lee, Kyung-Jong
Abstract:
DNA double-strand breaks (DSBs) are among the most lethal forms of DNA damage. The nonhomologous end-joining (NHEJ) pathway is the principal mechanism for repairing DSBs in mammalian cells. It is also required for V(D)J recombination. There are at least four essential proteins in this pathway. These include Ku protein, DNA PKcs, and the DNA Ligase IV/XRCC4 (DNL IV/XRCC4) complex. This dissertation reports the determination of the quaternary structure of the DNL IV/XRCC4 complex, the mapping of a major human autoimmune epitope in XRCC4, the identification of DNAPKcs phosphorylation sites in XRCC4, and an investigation of the biochemical significance of XRCC4 phosphorylation. Biochemical characterization shows that DNA Ligase IV and XRCC4 form a stable mixed heterotetramer. This is the active form of the enzyme and is essential for in vitro DNA end joining in the presence of additional factors derived from cell extracts. Data shown here also demonstrate that the DNL IV/XRCC4 complex is a human autoantigen. The major autoimmune epitope maps to amino acids 251-266. This epitope coincides with several sites where XRCC4 is potentially modified in response to radiation or inflammation, including a DNA-PKcs phosphorylation site at serine 260. Results raise the possibility that radiation-induced post-translational modifications contribute to development of an autoimmune response in susceptible individuals. Previous work has shown that DNA-PKcs kinase activity is required for NHEJ, but the critical physiological target of this enzyme is not yet known. Current work shows that DNA-PKcs phosphorylates serine 318 of XRCC4, in addition to the serine 260 site described above. The presence of serine 260 increases phosphorylation at serine 318, suggesting that phosphorylation can occur sequentially. Mutation o f serine 260 reduced DNA end-joining activity and sensitivity to the PI3 kinase inhibitor (LY294002). These data provide preliminary evidence that phosphorylation of XRCC4 by DNA-PKcs contributes to regulation of DNA repair.
Affiliation:
Institute of Molecular Medicine and Genetics
Issue Date:
Nov-2002
URI:
http://hdl.handle.net/10675.2/347146
Additional Links:
http://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/305464047?accountid=12365
Type:
Dissertation
Appears in Collections:
Theses and Dissertations

Full metadata record

DC FieldValue Language
dc.contributor.authorLee, Kyung-Jongen
dc.date.accessioned2015-03-25T21:59:20Zen
dc.date.available2015-03-25T21:59:20Zen
dc.date.issued2002-11en
dc.identifier.urihttp://hdl.handle.net/10675.2/347146en
dc.description.abstractDNA double-strand breaks (DSBs) are among the most lethal forms of DNA damage. The nonhomologous end-joining (NHEJ) pathway is the principal mechanism for repairing DSBs in mammalian cells. It is also required for V(D)J recombination. There are at least four essential proteins in this pathway. These include Ku protein, DNA PKcs, and the DNA Ligase IV/XRCC4 (DNL IV/XRCC4) complex. This dissertation reports the determination of the quaternary structure of the DNL IV/XRCC4 complex, the mapping of a major human autoimmune epitope in XRCC4, the identification of DNAPKcs phosphorylation sites in XRCC4, and an investigation of the biochemical significance of XRCC4 phosphorylation. Biochemical characterization shows that DNA Ligase IV and XRCC4 form a stable mixed heterotetramer. This is the active form of the enzyme and is essential for in vitro DNA end joining in the presence of additional factors derived from cell extracts. Data shown here also demonstrate that the DNL IV/XRCC4 complex is a human autoantigen. The major autoimmune epitope maps to amino acids 251-266. This epitope coincides with several sites where XRCC4 is potentially modified in response to radiation or inflammation, including a DNA-PKcs phosphorylation site at serine 260. Results raise the possibility that radiation-induced post-translational modifications contribute to development of an autoimmune response in susceptible individuals. Previous work has shown that DNA-PKcs kinase activity is required for NHEJ, but the critical physiological target of this enzyme is not yet known. Current work shows that DNA-PKcs phosphorylates serine 318 of XRCC4, in addition to the serine 260 site described above. The presence of serine 260 increases phosphorylation at serine 318, suggesting that phosphorylation can occur sequentially. Mutation o f serine 260 reduced DNA end-joining activity and sensitivity to the PI3 kinase inhibitor (LY294002). These data provide preliminary evidence that phosphorylation of XRCC4 by DNA-PKcs contributes to regulation of DNA repair.en
dc.relation.urlhttp://ezproxy.gru.edu/login?url=http://search.proquest.com/docview/305464047?accountid=12365en
dc.rightsCopyright protected. Unauthorized reproduction or use beyond the exceptions granted by the Fair Use clause of U.S. Copyright law may violate federal law.en
dc.subjectNon-homologous End Joiningen
dc.subjectDNA Ligase IVen
dc.subjectXRCC4en
dc.subjectDNA-PKcsen
dc.subjecthuman autoanitgenen
dc.titleCharacterization o f the DNA Ligase IV and XRCC4 complex in the DNA double-strand break repairen
dc.typeDissertationen
dc.contributor.departmentInstitute of Molecular Medicine and Geneticsen
dc.description.advisorDynan, William S.en
dc.description.committeeTuan, Dorothy Y. H.; Mivechi, Nahid F.; Pollock, Jennifer S.; Takeda, Yoshihikoen
dc.description.degreeDoctor of Philosophy (Ph.D.)en
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