Maintaining DNA Replication Fork Stability:  Role of the Fanconi Anemia Pathway (2009 - Present)

Investigator:  Niall Howlett, University of Rhode Island
Mentor:  Ralph Scully, Beth Israel Deaconess Hospital and Harvard University 

Abstract: Fanconi anemia (FA) is a recessive disorder characterized by congenital abnormalities, progressive bone marrow failure, and pronounced cancer susceptibility. The FA pathway is required for the cellular response to DNA crosslinking agents, e.g. mitomycin C. The in vivo physiological function of the FA pathway, however, remains unknown. We have recently made the important observation that the FA pathway is functionally required for the cellular response to disruption of DNA replication. The FA pathway is strongly activated via the mono-ubiquitination of the FANCD2 protein following treatment with the DNA polymerase inhibitor aphidicolin (APH). Abrogation of the FA pathway leads to increased chromosome breakage, including breakage at the common chromosomal fragile sites FRA3B and FRA16D, following treatment with APH. We hypothesize that the FA pathway plays an integral role in the maintenance of DNA replication fork stability, a function critical for the prevention of neoplastic transformation and cancer. Two aims are proposed to gain further insight into the role of the FA pathway in the maintenance of DNA replication fork stability. First, we will characterize the interaction between the FANCD2 protein and the major DNA polymerase processivity factor PCNA. Using site-directed mutagenesis we will mutate the critical amino acid residues of two recently identified FANCD2 PCNA interaction-motifs and determine the functional significance of the FANCD2-PCNA interaction. Second, using integration reporter plasmids, we will determine if the increased DNA replication stress-induced chromosome breakage of FA cells leads to the increased integration of foreign DNA in vitro. Finally, we will attempt to establish a clinical corollary for a role of the FA pathway in the in the maintenance of DNA replication fork stability: We will determine if the increased common chromosomal fragile site breakage of FA cells is associated with an increased frequency of human papillomavirus (HPV) DNA integration at these loci, by mapping the genomic sites of HPV integration in FA tumor tissue. Our proposed studies may lead to new findings regarding the in vivo physiological role of the FA pathway, and provide important insight into the increased cancer susceptibility of FA patients.