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Pathology and Laboratory Medicine
B.M.D., M.S., 2000, Tianjin Medical University, P. R. China
Thesis Advisor: Muriel W. Lambert, Ph.D.
Department: Pathology and Laboratory Medicine
Friday, July 25, 2008
Repair of DNA interstrand cross-links (ICLs) is a critical process, and failure to repair this type of damage can result in errors in transcription and DNA replication which in turn can lead to chromosomal abnormalities, mutagenesis and cell death. Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, diverse congenital abnormalities, genomic instability and a marked predisposition to cancer. There are thirteen FA complementation groups and cells from all these FA patients show a marked hypersensitivity to ICL agents and are deficient in ability to repair this type of damage. Repair of ICLs is a multi-step process that involves damage recognition and incision, translesion DNA synthesis (TLS), homologous recombination (HR) and nucleotide excision repair (NER). Our laboratory has shown that the structural protein, nonerythroid á spectrin (áSpII), is involved in the initial damage recognition step of the repair process and possibly some of the subsequent steps. The DNA repair protein, ERCC1-XPF, has also been shown to be involved in this initial step as have several of the FA proteins. We have hypothesized that áSpII, ERCC1-XPF, and some of the FA proteins play a role in these initial steps. In the present study, the role of FA proteins in the repair process was further examined, in particular the interactions of several of the FA core complex proteins, FANCA, FANCC, FANCF and FANCG, with proteins involved in the initial steps of ICL repair. We have previously shown that FANCG binds directly to áSpII. Yeast two-hybrid analysis has now been undertaken to examine the interaction of other FA core proteins with áSpII and ERCC1-XPF and, in addition, to examine the interaction of ERCC1-XPF with áSpII. Four overlapping regions of áSpII were constructed. It was found that of the FA proteins only FANCG bound to áSpII. ERCC1-XPF did not bind directly to áSpII. Of the FA proteins examined, only FANCG bound to ERCC1 and XPF. This binding was particularly strong between FANCG and ERCC1. Analysis of the domains involved in this interaction showed that the central domain of ERCC1 (residues 120-220) and the C-terminal region of FANCG (residues 263-622) were sufficient for this interaction. Since FANCG contains seven tetratricopeptide repeats (TPRs), which are important protein-protein interaction motifs, site-directed mutagenesis studies were undertaken to evaluate the importance of each individual TPR motif for the binding to ERCC1. It was found that TPR1, 3, 5 and 6 are essential for the binding of FANCG to ERCC1. Mutation in TPR2 affected binding to a lesser degree. TPR7 was not important at all for the interaction. These studies thus demonstrate that there is a direct link between an FA protein, FANCG, and proteins involved in the initial damage recognition/incision steps in the cross-link repair process. FA proteins may, therefore, be an important part of the ICL repair machinery and a deficiency in these proteins, as occurs in FA, could have serious consequence