Post-translational modification (PTM) constitutes a ubiquitous mechanism to expand proteins' structure, interactions, localization, and function. The research covered in this dissertation focuses on the identification and characterization of the functional roles of the novel PTMs of damaged DNA-binding protein 2 (DDB2) and MRG15, which are important in nucleotide excision repair (NER) and homologous recombination (HR), respectively.
In Chapters two and three, by employing LC-MS/MS, I discovered, for the first time, that DDB2 could be methylated on the α-amino group of N-terminal alanine (after cleavage of the initiating methionine) and phosphorylated at serine 26 in HEK293T human embryonic kidney epithelial cells. In addition, this α-N-methylation was found to be catalyzed by the N-terminal Xaa-Pro-Lys N-methyltransferase 1 (NTMT1). I also observed that the level of serine 26 phosphorylation was significantly reduced in cells treated with flavopiridol, an inhibitor for cyclin-dependent kinases (Cdks), suggesting the involvement of Cdks in this phosphorylation.
The importance of this α-N-methylation and Ser26 phosphorylation of DDB2 is illustrated by the observation that methylation-defective mutant of DDB2 displayed a reduced efficiency, while DDB2 mutant deficient in Ser26 phosphorylation failed, to be recruited to ultraviolet (UV) light-induced cyclobutane pyrimidine dimer (CPD) foci. Moreover, while Ser26 phosphorylation was also demonstrated to crosstalk with DDB2 ubiquitination and critical for proteasomal degradation of DDB2, loss of α-N-methylation or Ser26 phosphorylation of DDB2 conferred reduced ATM activation, decreased efficiency in CPD repair, and elevated sensitivity of cells toward UV light exposure. Collectively, I concluded that α-N-methylation and the phosphorylation of Ser26 in DDB2 plays a significant role in NER. This study also expanded the biological functions of protein α-N-methylation to DNA repair.
In Chapter four, I further expanded the targets of NTMT1 in human cells to MRG15. Importantly, I demonstrated, for the first time, the involvement of α-N-methylation in protein-protein interaction. My results showed that the α-N-methylated N-terminus of MRG15 enables its interaction with TIP60 histone acetyltransferase through binding to the chromo domain of TIP60 and stimulates allosterically the enzymatic activity of the latter. In addition, I found that this α-N-methylation-chromo domain interaction is indispensable for the acetylation of lysine 16 in histone H4 (H4K16Ac), for DNA damage-induced ATM activation, for the repair of DNA double strand breaks via the homologous recombination pathway, and for protecting cells from the genotoxic effects of ionizing radiation and interstrand cross-linking agents. Moreover, I defined MRG15 as a molecular determinant for a novel histone crosstalk between histone H3 lysine 36 trimethylation (H3K36me3) and H4K16Ac. Together, my study unveiled, for the first time, the α-N-methylation of MRG15 and discovered novel functions of protein α-N-methylation in the context of DNA damage response and repair. I also uncovered a novel trans-histone modification where H3K36me3 drives H4K16Ac in human cells, revealed the essential role of MRG15 in this process, and demonstrated the importance of this trans-histone modification in ATM activation and homologous recombination repair.