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Abstract
Non-homologous end joining (NHEJ) and homologous recombination (HR) are the two main DNA double-strand breaks (DSBs) repair pathways utilized to maintain genome stability in mammalian cells. NHEJ is initiated by binding of the Ku70/86 heterodimer, which subsequently recruits the DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) protein (alternately referred to as PRKDC, protein kinase, DNA-dependent, catalytic subunit) to form the trimeric DNA-PK complex. After limited end processing by Artemis (a nuclease) and DNA polymerases Pol λ and Pol μ, the broken ends are re-ligated by a complex consisting of DNA ligase IV, XRCC4 (X-ray cross complementing 4) and XLF (XRCC4-like factor). The mechanism of NHEJ has been studied for decades, but in spite of that, the role and importance of DNA-PKcs in NHEJ remains unclear. To illuminate the function of DNA-PKcs in NHEJ, our laboratory has generated two human colorectal carcinoma HCT116 cell lines that are DNA-PKcs-deficient: a complete knockout (DNA-PKcs-/-) and a version expressing a dominant-negative kinase-dead (KD) protein from one allele (DNA-PKcsKD/-). The latter cell line will be featured prominently in this thesis.
In contrast to NHEJ, HR requires the displacement of Ku70/86 heterodimers from the damaged DNA ends to instead facilitate end resection by Meiotic recombination defective 11 (Mre11):Radiation sensitive 50 (Rad50):Nijmegen chromosome breakage syndrome 1 (NBS1) commonly referred to as the MRN complex. MRN is often assisted in this resection process by the C-terminal binding protein 1 (CtBP1) interacting protein (CtIP). This initial resection is then enhanced by additional nucleases, primarily DNA replication helicase/nuclease 2 (DNA2), and Exonuclease 1 (EXO1). Human replication protein A (RPA) and Radiation sensitive 51 (RAD51) then bind to the 3’ overhangs generated by end resection to sequentially protect ssDNA and perform the strand invasion process, respectively. Depending on whether a double Holliday junction is formed, HR is divided into two sub-pathways: double-strand break repair (DSBR) and synthesis-dependent strand annealing (SDSA). These pathways can then facilitate essentially error-free repair resulting in products with (DSBR) or without (DSBR and SDSA) crossovers.
In mammals, NHEJ- or HR-mediated repair results in radically different repair products and this fact underscores the importance of a cell making the proper pathway choice decision when a DSB happens. How pathway choice is mechanistically decided, however, is unclear. Parsimoniously, it seems likely that there will be regulators (both positive and negative) for both pathways and that the interplay and or varied expression of these regulators will modulate pathway choice. To this end, after the core repair proteins that are required for the NHEJ and HR pathways had been identified, recent research has focused on the impact of chromatin status on DSB repair pathway choice. In particular, histone acetylation and its “reader” proteins — bromodomain-containing proteins — have been shown to play central roles in regulating chromatin status and through this to influence both the efficiency and pathway choice of DSB repair.
The goal of this thesis work was to identify and then characterize novel DSB repair regulators. Multiplexed inhibitor beads (MIBs) coupled mass spectrometry (MS) was performed to determine the kinome change in a DNA-PKcsKD/- cell line. MIBs consist of multiple covalently bound ATP analogs. Therefore, proteins with ATP binding pockets will be captured by MIBs and can be subjected to MS analysis. The rationale for this approach was that since NHEJ is ablated in this cell line the cells may need to upregulate HR in order to survive and we postulated that some of these regulators might be ATP-binding or utilizing proteins. Through MIBs-MS, I have identified Bromodomain containing protein 3 (BRD3), a member of bromodomain and extra-terminal domain (BET) family, as a novel DSB repair regulator.
