Zach Rodriguez

University of California, Santa Barbara

Investigating the Interaction Between FANCD2 and Extrachromosomal DNA in the Context of Gene Editing

Investigating the Interaction Between FANCD2 and Extrachromosomal DNA in the Context of Gene Editing

Zach Rodriguez, Christine Joyce, Amy Wang, Chris Richardson

Double strand breaks (DSBs), a particularly lethal type of DNA damage, can be repaired by eukaryotic cells via non-homologous end-joining (NHEJ) or homology-directed repair (HDR). NHEJ is dependent on an error prone ligation event that often causes insertions or deletions of nucleotides. HDR utilizes a homologous template such as a sister chromatid, homologous chromosome, or extrachromosomal DNA with homology and results in perfect repair at a much higher rate than NHEJ. HDR is also the molecular mechanism that is responsible for facilitating CRISPR-Cas9 gene editing. Previous work by our lab identified that the Fanconi Anemia (FA) pathway impacts DNA repair outcomes, and that a key FA pathway protein, FANCD2, stabilizes the extrachromosomal template molecules used in gene editing. Our objective in this study was to determine the properties of FANCD2 that allow it to stabilize extrachromosomal DNA. To do this we enlisted two different approaches: (1) small molecule inhibitors and (2) genetic mutants. First, we show that FACND2 promotes transgene expression. We then show that this transgene expression is dependent on FANCD2 ubiquitination and independent of FANCD2 deubiquitination. Lastly, we show that this expression is increased by treatment with a small molecule inhibitor that has been previously characterized for its ability to prevent FANCD2 from binding to damaged chromosomal DNA. The results from these experiments will lead to a greater understanding of how FANCD2 interacts with extrachromosomal DNA. Understanding how eukaryotic cells interact with extrachromosomal template molecules may lead to improvements in gene editing.

NIH UC Santa Barbara Center for Science and Engineering Partnerships UCSB California NanoSystems Institute MCDB