Nucleotide excision repair (NER) plays an important role in maintaining genomic integrity through removing helix-distorting DNA damages caused by UV irradiation or chemical mutagens. Defects in NER underlie the human hereditary disease, xeroderma pigmentosum, which is characterized as sensitivity to ultraviolet light and a high incidence of skin cancer. Studies of NER have been focused on identifying components and the biochemistry of excision and repair reactions, while little is known about the regulatory mechanisms cells employ to control the NER activity. The Cullin 4A ubiquitination machinery has recently been shown to mediate ubiquitin-dependent proteolysis of the p48 subunit of damaged DNA binding proteins that are believed to participate in the initial DNA damage recognition step of NER. Our long-range goal is to understand how the ubiquitin-proteolytic pathway regulates NER, and to relate this understanding to human diseases associated with defective NER as well as the malfunctions of the ubiquitination machinery. The objective of this application is to understand the molecular basis, the regulatory pathways, and the functional significance of CUL-4A-mediated p48 degradation in controlling the damage-sensing step of nucleotide excision repair. The central hypothesis of the application is that the CUL-4A ubiquitination machinery controls the ability of the NER machinery to recognize and remove specific DNA damages through restricting the abundance of p48.
The specific aims proposed are (1) To determine the molecular basis for CUL- 4A/DDB interactions and for subcellular distribution of DDB proteins. (2) To determine the functional significance of CUL-4A in DNA damage recognition and repair. (3) To assess the role of c-Abl in regulating p48 degradation and nucleotide excision repair. The results of this work will provide a new paradigm for the regulation of nucleotide excision repair by ubiquitin-dependent proteolysis, and generate a better understanding of the biochemical mechanisms controlling the intracellular distribution and abundance of DDB proteins. Completion of the proposed studies will also shed light on how abnormal activation of CUL- 4A contributes to tumor development.
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