The Genetic Dissection of Shieldin

Abstract

Multiple DNA repair pathways have evolved over the course of cell evolution. Homology-directed repair (HDR) and non-homologous end joining (NHEJ) are two known pathways that can act in an antagonistic manner when facing DNA double-stranded breaks (DSBs). BRCA1 and 53BP1 are DNA repair proteins of the HDR and NHEJ pathways, respectively. In the event of BRCA1 mutation, HDR is rendered defective, allowing the accumulation of carcinogenic mutations that lead to PARPi-sensitive breast and ovarian cancers. PARPi chemotherapeutic drug toxicity in BRCA1-deficient cancer cells is mediated by 53BP1, which in addition to its role in NHEJ, suppresses HDR. Here, we set out to investigate the genetics of shieldin, a four-subunit protein complex downstream of 53BP1 in NHEJ. Specifically, the gene interaction type between individual shieldin subunits, as it relates to their role in HDR suppression, is probed. It is hypothesized that each subunit has a distinct role in contributing to shieldin function, and hence necessary for HDR suppression. The approach involves utilization of CRISPR-Cas9 technology, chemotherapeutic drug sensitivity, and RAD51 focus formation assays. Altogether, the present study aims to assess the mechanistic basis underlying shieldin function and contribution to HDR suppression in PARPi-sensitive BRCA1-deficient cells.