Functional and Structural Study of Protein-protein Interactions in Nucleotide Excision Repair

Abstract

Nucleotide excision repair (NER) is the main repair pathway that removes bulky adducts induced by UV irradiation, chemotherapeutic reagents and environmental sources from DNA. People with defects in NER suffer from xeroderma pigmentosum (XP) and are highly sensitive to UV light and show a 2000-fold increased incidence of skin cancer. NER is categorized into two sub-pathways in damage recognition step: global-genome (GG)-NER and transcription-coupled (TC)-NER. In GG-NER, XPC-RAD23B-CETN2 recognizes DNA damage and recruits TFIIH to open the DNA. In TC-NER, stalled RNA polymerase II recruits TFIIH with the help of CSB, CSA, UVSSA, ELOF1 and STK19. After damage verification by TFIIH, two sub-pathways merge to generate preincision complex with the involvement of XPA, RPA, and XPG. Finally, ERCC1-XPF joins the complex and incises the DNA 5’ to the lesion, whereas XPG performs a 3’ incision to the lesion. After incision by endonucleases, the replication machinery fills the gap and a DNA ligase seals the nick to complete NER. In Chapter 1, we review the protein-protein interactions between the core NER factors. Here, we highlight how the study of interactions among proteins by structural and/or functional studies have provided insight into the NER machinery. From damage recognition to damage excision, we discuss how protein-protein interaction among NER factors coordinate the activities in the pathway. In Chapter 2, we show that XPA interactions with two RPA subunits, RPA70 and RPA32 contribute synergistically to NER. We found that defects of both interactions affect NER activity and that abolishing the XPA-RPA32 interaction affects complex assembly, while the XPA-RPA70 interaction is needed for completion of NER. Based on our SAXS analysis of the XPA-RPA-DNA interaction, we were able to propose a model of NER preincision complex showing that DNA has a compact U shape in the complex that is held in place by the XPA-RPA70 interaction. Altogether, this work showed how XPA-RPA interactions organize the preincision complex. In Chapter 3, studying NER function through XP patient variants, we studied an XPA allele with a mutation in the TFIIH interaction domain. XPA H244R patients present mild symptoms of XP without skin cancer, but suffer from marked neurological features, including cerebellar ataxia. We show that XPA-H244R has a severely weakened interaction with the TFIIH complex leading to a reduced association for downstream endonuclease ERCC1-XPF with NER complexes. Interestingly, despite these defects, cells expressing XPA-H244R show considerable levels of residual GG-NER (~50%). By contrast, XPA-H244R is fully deficient in TC-NER, thus representing the first know XPA allele with a separation of function mutation for GG- and TC-NER. Our characterization of an XPA mutation that interferes with TFIIH binding showed importance of C-terminal of XPA especially for TC-NER. To get a more complete picture of protein-protein interactions in the NER preincision complex, we studied the interaction of TFIIH with XPG in Chapter 4. After damage verification by TFIIH, XPG joins the preincision complex and incises the DNA 3’ to the damage. It is unknown how XPG interacts with TFIIH to enable NER. Based on sequence alignment and computational modeling, we were able to predict several regions in XPG to interact with the p62 and XPD subunits of TFIIH. Cells expressing XPG with mutations in the p62 or XPD interaction regions were sensitive to UV irradiation and showed reduced rates of NER. Mutations in p62 binding domain of XPG had a moderate effect on NER activity, while mutations in XPD binding domain result in an almost complete NER defect. This study provides insights into how TFIIH interacts with XPG for the formation of the preincision complex and execution of dual incision.