MAINTAINING DNA REPLICATION FORK STABILITY: ROLE OF THE FANCONI ANEMIA PATHWAY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Fanconi anemia (FA) is a rare recessive disorder characterized by bone marrow failure and pronounced cancer susceptibility. The FA pathway is required for the cellular response to DNA cross-linking agents, e.g. mitomycin C. However, the in vivo physiological function of the FA pathway remains unknown. We have recently discovered 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 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 cancer. Two aims are proposed to gain further insight into this role. First, we will characterize the interaction between FANCD2 and the 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 increased integration of foreign DNA. Finally, we will attempt to establish a clinical corollary for this phenotype: We will determine if the increased 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 head and neck cancers. Our proposed studies will lead to new findings regarding the in vivo physiological role of the FA pathway, and provide insight into the increased cancer susceptibility of FA patients.