Date of Graduation
5-2014
Document Type
Dissertation (PhD)
Program Affiliation
Biomedical Sciences
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Lei Li
Committee Member
Junjie Chen
Committee Member
Randy J Legerski
Committee Member
Bin Wang
Committee Member
Pierre D McCrea
Abstract
Cells derived from Fanconi anemia (FA) patients are characterized by hypersensitivity to DNA interstrand crosslinks (ICLs), suggesting that FA genes play a role in ICL repair. Fanconi anemia core complex (including A, B, C, E, F, G, L, FAAP20, and FAAP100) activates the Fanconi pathway by providing the essential E3 ligase activity for FANCD2 mono-ubiquitination. Previous studies suggested the existence of three protein-protein interaction groups. However, the functions of most FA core complex protein are still limited to their presence in the complex. How the spatially-defined FANCD2 ubiquitination is accomplished by the core complex remains unknown.
To elucidate the roles of FA core complex proteins in ICL response, especially their contribution to FANCD2 ubiquitination, we established isogenic knockout mutants deficient in FA core genes and determined the loss-of-function effects on the activation of FA pathway and on cellular survival against ICLs. Our results suggest three potential functional modules in the FA core complex: FANCB-FANCL-FAAP100 (B-L-100), FANCA-FANCG-FAAP20 (A-G-20), and FANC-FANCE-FANCF (C-E-F). We showed that the B-L-100 sub complex is the catalytic module absolutely required for the E3 ligase function of FA core complex. The A-G-20 module anchors the catalytic module to the chromatin. The C-E-F module acts as a loading factor of the core complex through FANCM. Our work revealed that the FA core complex is assembled with functional modules and carries out the spatially-defined FANCD2 monoubiquitination reaction to ensure that nucleases for DNA damage processing are enriched at the site of lesion. The deviate functions of different FA core complementation groups imply differential disease biology related to prognosis and treatment.
Keywords
Fanconi anemia, interstrand crosslink, DNA repair