UC Riverside

DNA is exposed to relentless challenges by a variety of chemical, enzymatic and environmental agents. Maintaining the integrity of the genome by immediate and precise repair mechanisms is one of the most efficient processes inside cells. The nucleotide excision repair pathway (NER) is one of the most important repair mechanisms used by the cells to remove bulky DNA lesions specifically generated by exposure to UV, ionizing radiations and chemotherapeutic agents like cisplatin. Several proteins work simultaneously to operate this pathway including, TFIIH multiprotein complex containing ten subunits and ERCC1-XPF. Several studies in the past have proposed that p53 protein, product of a tumor suppressor gene, plays key role in regulating the process of DNA repair. It has been shown to modulate the activity of multiple proteins involved in NER either by regulating their transcription or directly binding to them. However, as this process is very complex and well regulated, deciphering the mechanism of action has been a challenge.

In this thesis, we have attempted to gain some more insight into the role of p53 in NER by identifying proteins whose activity might be directly influenced by it. The two proteins that we have primarily concentrated on includes XPB protein and ERCC1-XPF complex. XPB, the largest subunit of TFIIH complex, uses its ATPase and unidirectional 3’-5’ helicase activities in accordance with the 5’-3’ helicase activity of XPD (another subunit of TFIIH) in NER to unwind the DNA helix near lesions. ERCC1-XPF is a structure specific endonuclease that cleaves the damaged DNA at 5’ end of lesion for subsequent NER factors to fill in the gap with correct nucleotide sequence, complementary to the template. We employ in vitro and in vivo techniques to explore the interaction of XPB and ERCC1-XPF with p53 protein and map down the specific modules of each protein important for this interaction.

In Chapter 1, we introduce the fundamental reasons behind DNA damage and different DNA repair pathways responsible for maintaining the genome. We also provide an overview of the process of NER along with detailed description about XPB DNA helicase, ERCC1-XPF endonuclease complex and p53 protein. This is followed by a discussion about the role of p53 in DNA repair. We then outline the principle of all the techniques used in this study to explore protein-protein interactions.

In Chapter 2, we provide evidence confirming that XPB could be one of the factors responsible for the interaction between p53 and TFIIH complex, regulating the DNA repair system. Firstly, we established this interaction using affinity column chromatography for pull-down assay and Fluorescence Resonance Energy Transfer (FRET). Further we utilized yeast two hybrid analysis to illustrate that smaller modules of XPB including 105-129 a.a at the N-terminal and 730-782 a.a at the extreme C-terminal bind to DNA binding domain (DBD) and C-terminal domain (CTD) of XPB. We also demonstrate that XPB-p53 protein-protein interaction is weakened in case of T119P mutation while is completely abolished in case of F99S and XPB11BE mutations respectively.

In Chapter 3, we mapped down the domains of interaction between ERCC1-XPF complex and p53. We provide results from yeast two hybrid analysis indicating that XPF subunit of ERCC1-XPF complex does not interact with p53. In contrast, we also illustrate that ERCC1 bind to DNA binding domain (DBD) and C-terminal domain (CTD) domain of p53 primarily with its central domain. However, the helix-hairpin-helix (HhH2) domain of ERCC1 might also be play a critical role in this interaction.

Finally, a summary of the major developments made in this thesis along with a discussion about the future direction is presented in Chapter 4.