Linking The Multiple Functions of XPF-ERCC1 Endonuclease in DNA Repair to Health Outcomes: Cancer and Aging

[摘要] XPF-ERCC1 is a structure specific endonuclease in which the XPF subunit is involved in nucleolytic activity and the ERCC1 subunit is involved in DNA binding. They are essential for multiple genome maintenance mechanisms which include the repair of bulky DNA monoadducts via nucleotide excision repair (NER) and also the repair of DNA interstrand crosslinks.In humans, the deficiency of XPF-ERCC1 results in two major syndromes: Xeroderma pigmentosum (XP), characterized by predisposition to skin cancer and XFE, characterized by symptoms of premature aging. The mechanism behind XP has been attributed to the inability to carry out NER. However, the mechanism behind the contribution of XPF-ERCC1 deficiency to aging has not yet been established.The most successful chemotherapeutic agents are crosslinking agents that cause both monoadducts and interstrand crosslinks in DNA. Unfortunately, ~50% of tumors are resistant to these drugs. Numerous studies implicate XPF-ERCC1 as a major culprit in tumor resistance because of its pivotal role in repairing DNA damage but to date, there is no conclusive evidence of this. This dissertation aims to advance our understanding of the mechanistic contribution of XPF-ERCC1 to two major issues of public health relevance, cancer and aging. The first chapter examines the importance of DNA binding residues of XPF-ERCC1 located in the c-terminus of both proteins. The results revealed that mutations in DNA binding residues located in the HhH domain of ERCC1 and the nuclease domain of XPF have a deleterious effect on nuclease and NER activity. Analysis of the n-terminus of XPF revealed that mutations in the n-terminus confer selective crosslink sensitivity to cells thereby identifying target regions of the XPF gene that could be exploited to generate an ICL repair pathway specific defect. To probe the functionality of XPF-ERCC1 as biomarkers of chemoresistance, we developed a novel monoclonal antibody, 1E4, which could be used to establish a positive correlation between XPF-ERCC1 protein levels and patient sensitivity to crosslinking regimes. To examine whether DNA repair capacity of an individual can predict lifespan, and to determine a way to diagnose disorders such as XP, we developed a novel semi-automated assay to measure the NER capacity of suspension cells.

[发布机构] the University of Pittsburgh