The Effect of FANCL on Recruitment of FANCD2 to Double Strand Breaks

DNA damage often arises from normal cellular processes, such as cellular respiration, or from damage outside the cell, such as ionizing radiation or chemotherapy drugs. The most severe type of DNA lesions are double strand breaks, or DSBs, which can cause cell death, chromosomal abnormalities, or cancer. Cells implement DNA repair pathways in response to this damage. The two main repair mechanisms are non-homologous end-joining (NHEJ), which can produce insertions or deletions, or homology-directed repair (HDR), which uses a template strand to result in perfect repair. HDR is facilitated by the Fanconi Anemia (FA) pathway. FANCD2 is a protein within the FA pathway that localizes to DSBs during DNA repair, but the requirements for FANCD2 localization are currently unknown. The goal of this project is to determine if a component of the FA core complex, FANCL, is needed for FANCD2 recruitment to DSBs. Our methodology consists of creating DSBs using a CRISPR/Cas9 system within a FANCL knockdown cell line to wild-type K562 cells. We will compare the amount and localization of FANCD2 on the cell lines using chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR). Our preliminary results have shown lower cell survival in the FANCL knockdown cell line, which indicates that this component could possibly aid in DNA repair. Understanding DNA repair can improve traditional cancer treatments, as well as enhance gene editing.