Investigation Into the Role of BAP1 in DNA Damage and Repair

Abstract

The DNA of living organisms is constantly experiencing wide range of DNA damaging agents that can result in altered DNA bases or more serious damage such as double-strand breaks (DSBs). Unrepaired DNA damage can lead to genome instability and it is implicated in disease process including cancer.TP53 binding-protein 1 (53BP1) is a key DSB repair protein that regulates the repair pathway choice1. It is implicated in PARP inhibitor resistance in BRCA1-dificient tumors2,3. In order to identify novel 53BP1 regulators, we performed a screen using an siRNA library against the human deubiquitinating (DUBs) enzymes with 53BP1 ionizing radiation induced foci as a read out. Two DUBs achieved a top negative score; BRCA1 Associated protein 1 (BAP1). BAP1 is an important tumor suppressor protein whose function is lost in more than 7% of all cancers4. Here we demonstrate that BAP1 stabilizes 53BP1 by preventing its ubiquitination and degradation by the proteasome. BAP1 associates with 53BP1 and it is required for 53BP1 ionizing radiation (IR)-induced foci (IRIF) formation, G2/M checkpoint and radiation sensitivity. Similar to 53BP1, BAP1 deficiency confers PARP inhibitor resistance in BRCA1-deficient cells, and it leads to impaired immunoglobulin class switch recombination. Additionally, we discovered a ubiquitin ligase, UBE2O interaction with 53BP1 diminished after IR damage, and overexpression of UBE2O leads to diminished 53BP1 IRIF formation. We show evidence that BAP1 and UBE2O binds 53BP1 nuclear localization signal (NLS) basic stretch within its minimal focus forming region (FFR), and they regulate FFR ubiquitination and recruitment to the histone basic patch. Through further work, we discovered that an NLS binding family (Karyopherin (KPNA) family) interacts with and regulates 53BP1 recruitment. Our findings shed light into important aspects of 53BP1 regulation through deubiquitination and provide mechanistic insights into the function of an important tumor suppressor BAP1.