Nucleotide excision repair (NER) is the major DNA repair machinery that removes DNA damage induced by ultraviolet light (UV) and chemical mutagens to prevent genomic instability and tumorigenesis. While the enzymatic reactions for excision and repair of DNA photolesions are well studied, regulatory pathways governing the temporal and spatial control of DNA damage recognition remains poorly understood, and the physiological functions of such regulation on tumor suppression have not been explored due to the unavailability of animal models. The cullin 4A (CUL-4A) ubiquitin ligase has recently emerged as a key regulator of two DNA damage sensors: the damaged DNA binding proteins (DDBs, heterodimers of DDB1 and DDB2) and xeroderma pigmentosum complementation group C (XPC) protein. Interestingly, recent studies revealed a second function of DDBs as integral components of the CUL-4A ubiquitin ligase complex. During the previous funding period, our biochemical and structural biology studies provided mechanistic insight into the assembly of the CUL-4A-DDB complex, and a novel kinase-independent function of c-Abl in activating CUL-4A-dependent ubiquitination of DDBs both under normal conditions and upon UV irradiation. Importantly, we generated conditional CUL-4A knockout mice and showed that skin-specific CUL-4A knockout mice were resistant to UV-induced skin carcinogenesis, suggesting an intriguing possibility of pharmacological inhibition of CUL-4A as a prevention strategy for UV-induced skin cancer. While CUL-4B shares overlapping functions with CUL-4A in cell growth and survival, its role on DDB2 degradation and NER appears less pronounced than that of CUL-4A. We also collaborated with Dr. Stephan Goff to determine the physiological functions of DDB1 in NER and in controlling cell cycle and genomic integrity in the conditional DDB1 knockout mice. Interestingly, our in vivo studies revealed dramatic upregulation of the cyclin-dependent kinase inhibitor p21/CIP1/WAF1 in CUL-4A-/- and DDB1-/- mice, as well as in MEF cells and keratinocytes derived from these mice. Our long-term goal is to understand how the ubiquitin pathway regulates DNA repair and affects tumor development. We hypothesize that the CUL-4A and CUL-4B ubiquitin ligase activity is precisely controlled both to ensure proper execution of NER and to halt cell cycle events to allow time for efficient repair. We are uniquely positioned to test this hypothesis since we have generated specific ubiquitination-resistant DDB2 mutants, identified a novel modulator (BRAP2) in the temporal control of CUL-4A activity following UV irradiation, and have CUL-4A, DDB1 and p21 (or CIP1 or WAF1) knockout mice in hand. We propose to employ a combination of biochemical, genetic and cell biological approaches to address the following three specific aims: (1) establish the mechanism by which DDB2 ubiquitination regulates damage recognition and repair;(2) To determine the temporal control of CUL-4A and CUL-4B ubiquitin ligase activity by BRAP2 during NER;(3) To determine the mechanistic basis and functional significance of p21 accumulation in protecting CUL-4A-deficient mice against UV-induced carcinogenesis. Successful completion of these aims will significantly contribute to our understanding of the molecular and genetic basis of the ubiquitin-proteasome pathway in DNA repair and tumorigenesis. Knowledge gained from these efforts could be exploited to devise novel strategies for the prevention and/or treatment of UV- and chemical mutagen-induced skin cancer or skin-related disorders, and thus improve the health and well-being of humans.

Public Health Relevance

DNA damage contributes to the development of 80-90% of human cancers. Ubiquitin- dependent proteolysis of DNA repair proteins is crucial for modulating functions of DNA damage recognition and removal in both normal cells and cancer cells. This study will characterize the roles of cullin 4A ubiquitin ligase in controlling the stability of damage sensors of the nucleotide excision repair apparatus. Knowledge gained from these efforts could be exploited to devise novel strategies for the prevention and/or treatment of UV- and chemical mutagen-induced skin cancer or skin-related disorders, and thus improve the health and well-being of humans.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA098210-10
Application #
8449697
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Okano, Paul
Project Start
2002-12-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
10
Fiscal Year
2013
Total Cost
$301,361
Indirect Cost
$123,041
Name
Weill Medical College of Cornell University
Department
Pathology
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
D'Alfonso, Timothy M; Hannah, Jeffrey; Chen, Zhengming et al. (2014) Axl receptor tyrosine kinase expression in breast cancer. J Clin Pathol 67:690-6
Kong, F; Zhang, J; Li, Y et al. (2014) Engineering a single ubiquitin ligase for the selective degradation of all activated ErbB receptor tyrosine kinases. Oncogene 33:986-95
Malatesta, M; Peschiaroli, A; Memmi, E M et al. (2013) The Cul4A-DDB1 E3 ubiquitin ligase complex represses p73 transcriptional activity. Oncogene 32:4721-6
Hannah, Jeffrey; Zhou, Pengbo (2011) Maximizing target protein ablation by integration of RNAi and protein knockout. Cell Res 21:1152-4
Li, Wei; You, Liru; Cooper, Jonathan et al. (2010) Merlin/NF2 suppresses tumorigenesis by inhibiting the E3 ubiquitin ligase CRL4(DCAF1) in the nucleus. Cell 140:477-90
Lee, Jennifer; Zhou, Pengbo (2010) Cullins and cancer. Genes Cancer 1:690-9
Hannah, Jeffrey; Zhou, Pengbo (2009) Regulation of DNA damage response pathways by the cullin-RING ubiquitin ligases. DNA Repair (Amst) 8:536-43
Liu, Liren; Lee, Sharrell; Zhang, Jianxuan et al. (2009) CUL4A abrogation augments DNA damage response and protection against skin carcinogenesis. Mol Cell 34:451-60
Cang, Yong; Zhang, Jianxuan; Nicholas, Sally A et al. (2007) DDB1 is essential for genomic stability in developing epidermis. Proc Natl Acad Sci U S A 104:2733-7
Li, Ti; Chen, Xiujuan; Garbutt, Kenneth C et al. (2006) Structure of DDB1 in complex with a paramyxovirus V protein: viral hijack of a propeller cluster in ubiquitin ligase. Cell 124:105-17

Showing the most recent 10 out of 12 publications