Author ORCID Identifier
http://www.orcid.org/0000-0003-2241-5066
Date of Graduation
5-2017
Document Type
Dissertation (PhD)
Program Affiliation
Genes and Development
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Bin Wang
Committee Member
Pierre McCrea
Committee Member
Xiaobing Shi
Committee Member
Zahid Hussain Siddik
Committee Member
Grzegorz Ira
Abstract
Genomes of all organisms are continuously damaged by numerous exogenous and endogenous sources leading to different kinds of DNA lesions, which if not repaired efficiently may trigger wide-scale genomic instability, a hallmark of cancer development. To overcome this, cells have evolved a sophisticated sensory network called the DNA damage response (DDR) comprised of a large number of distinct protein complexes categorized as sensor, mediator, transducer and effector proteins that amplify the DNA damage signal and activate cell cycle checkpoint to initiate DNA repair or trigger apoptosis where the defect is beyond repair. This intricate signaling pathway is tightly regulated by modulating DDR factors recruitment, retention and dissociation from the sites of DNA damage in a spatiotemporal manner mediated by numerous reversible post-translational modification (PTMs) including phosphorylation, ubiquitination, SUMOylation, methylation, acetylation, poly(ADP-ribosyl)ation, and Neddylation. In this study, I examined the role of phosphorylation and ubiquitination in regulating the DDR signaling at the DNA damage sites.
DNA double-strand breaks triggers a phosphorylation-mediated signaling at the damage sites leading to histone ubiquitination in Lys63-linked manner that recruits BRCA1-A complex to the damage sites. The A complex is comprised of BRCA1, Rap80, NBA1, BRE, BRCC36 and the adaptor protein Abraxas, which has been shown previously to constitutively interact with BRCA1-BRCT (BRCA1 C- terminal) domain through its C-terminal phosphorylated S406 residue. In this study, we found that DNA damage-induced Abraxas phosphorylation at neighboring S404 residue induces stable BRCA1 dimerization through its BRCT domain. Both crystal structure and in vivo analysis confirmed that phosphorylation at Abraxas S404 residue is essential for stable BRCA1-BRCT dimer formation and mutation in the S404 residue leads to impaired accumulation of BRCA1 to damaged chromatin. In addition, we found two germline mutations in the BRCA1-BRCT dimerization interface disrupt stable dimer formation both in vitro and in vivo.
Although phosphorylation has been shown to be the major PTM at the DSB sites, over the last decade, ubiquitination has also emerged as a key regulatory player in the DDR. Irradiation (IR)-induced DNA damage catalyzes Lys63-linked polyubiquitination of histones, H2A and H2A.X that leads to accumulation of BRCA1- A complex to DSBs. In my second study, we sought to determine whether non- lys63-linked ubiquitination also exists at the DSBs regulating the DDR pathway. My findings indicate that along with Lys63-linked ubiquitination, chromatin-bound proteins are also modified with Lys11-linked polyubiquitination at DNA damage sites in an ATM-dependent manner by Ube2S/Ube2C E2 conjugating and RNF8 E3 ligase enzymes and deubiquitinated by OTUD7B (Cezanne) enzyme. I further showed that histones H2A and H2A.X is modified with Lys11-linked polyubiquitination in a DNA damage-dependent manner that is essential for inhibiting transcriptional silencing at proximity to DSB sites to maintain genomic stability. Overall, my findings provide insights into how post-translational modifications regulate DDR factors dynamics at DSB sites and play a crucial role in maintaining genomic integrity.
Keywords
Phosphorylation, Ubiquitination, DNA damage response, BRCA1, Abraxas, Transcription silencing, Ube2S, RNF8, Histone