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YH dissertation 140417.pdf (4.53 MB)

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Functions of BRCA1, 53BP1 and SUMO isoforms in DNA double-strand break repair in mammalian cells

Hu, Yiheng
http://rave.ohiolink.edu/etdc/view?acc_num=osu1397756848

Abstract Details

2014, Doctor of Philosophy, Ohio State University, Molecular, Cellular and Developmental Biology.
In this dissertation study, we have investigated the protein functions in DNA double-strand break (DSB) repair of three important factors, BRCA1, 53BP1 and SUMO isoforms, at levels of biochemical activity, protein dynamics and chromosomal DNA repair. Our work reveals novel mechanisms of these proteins functioning in response to DSB damage, hence providing insights of where and how they are actively involved in each subpathway of DSB repair. In the first part of our work, we studied BRCA1, a tumor suppressor important for the maintenance of genomic stability including centrosome control and DSB repair, and found that a putative enzymatic mutant of BRCA1— BRCA1(I26A), which had been thought to disrupt its E3 ligase activity, was still functional in the cellular processes of regulating centrosome number and homologous recombination-dependent DSB repair, thereby raising a question of whether I26A mutant is indeed inert. Reevaluation of the ubiquitination activity of this BRCA1(I26A) mutant revealed that it is an active E3 ubiquitin ligase when associated with the appropriate E2 factor. We then think that conclusions about the dispensability of the BRCA1-dependent enzymatic activity in various cellular processes should be reconsidered. Next we studied the unique function of 53BP1, a known NHEJ factor for DSB repair. We found that 53BP1 specifically promotes the error-free conservative-NHEJ (C-NHEJ) mechanism, dependent on its upstream recruiters RNF8 and RNF168. 53BP1 has no effect on the highly mutagenic and deletional alternative-NHEJ (Alt-NHEJ) pathway or on homology-directed repair (HDR), but it suppresses single-strand annealing (SSA). We discovered that the localization of 53BP1 at sites of DSBs is accompanied by its bulk removal from the nucleus except at sites of DNA damage. And the degradation of bulk 53BP1 upon DNA damage is due to each action of RNF8 and RNF168. Further, we showed that failure to degrade bulk 53BP1 results in the failure for its downstream effector RIF1 to localize appropriately to DNA damage sites. These data provide a novel mechanism of 53BP1 responding to DSB damage. In the third part of our study, we assessed SUMO isoforms in DSB repair. We identified that SUMO isoforms act differentially in DSB repair pathways: SUMO1 stimulates all four subpathways while SUMO2/3 is only required for C-NHEJ pathway. Strikingly, the single SUMO E2 enzyme, UBC9, was required for C-NHEJ but not for HR or Alt-NHEJ. And the conjugation-deficient SUMO1 mutant protein was competent for HR and Alt-NHEJ repair similar to the wild-type, but not for C-NHEJ. Our data together reveal a novel role of SUMO1 as a free protein, not a protein conjugate in homologous recombination and alternative-NHEJ. Overall, we have identified biochemical steps at which these factors are required for DSB repair, as well as novel regulatory mechanisms controlling the process.
Jeffrey Parvin (Advisor)
Altaf Wani (Committee Member)
Qianben Wang (Committee Member)
Robin wharton (Committee Member)
131 p.
Molecular Biology
Double-strand break repair; BRCA1; 53BP1; SUMO; RNF8; RNF168; ionizing radiation

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Citations

  • Hu, Y. (2014). Functions of BRCA1, 53BP1 and SUMO isoforms in DNA double-strand break repair in mammalian cells [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397756848

    APA Style (7th edition)

  • Hu, Yiheng. Functions of BRCA1, 53BP1 and SUMO isoforms in DNA double-strand break repair in mammalian cells . 2014. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1397756848.

    MLA Style (8th edition)

  • Hu, Yiheng. "Functions of BRCA1, 53BP1 and SUMO isoforms in DNA double-strand break repair in mammalian cells ." Doctoral dissertation, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397756848

    Chicago Manual of Style (17th edition)

osu1397756848
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This open access ETD is published by The Ohio State University and OhioLINK.