| Human cells respond to internal and environmental sources of DNA damage with a myriad of pathways that induce cell cycle arrest, regulate programmed cell death and physically repair the damaged DNA. Collectively, these pathways are referred to as the DNA damage response. Central to the DNA damage response is a group of related protein kinases that includes the DNA-dependent protein kinase (DNA-PK), Ataxia-Telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR). ATM and ATR initiate DNA damage-induced cell cycle arrest while DNA-PK participates in the repair of DNA double strand breaks (DSBs). Each of these protein kinases is also involved in the DNA damage-induced phosphorylation of replication protein A (RPA), which is involved in various aspects of DNA repair, replication and recombination. Here we show using phosphospecific antibodies that the 32-kDa subunit of RPA (RPA32) is phosphorylated by both DNA-PK and ATM on Ser8, Thr21, and Ser 33 in vitro. In addition, we show that RPA32 is phosphorylated on various sites in human cells treated with DNA damaging agents such as ionizing radiation (IR), camptothecin (CPT) and etoposide (ETOP). The IR, CPT and ETOP-induced phosphorylation of RPA32 in vivo was dependent on ATM, DNA-PK and ATR, respectively. Furthermore, Thr21-phosphorylated RPA32 localizes to discreet regions within the nucleus in response to treatment with CPT. Based on the fact that three protein kinases separately participate in the DNA damage-induced phosphorylation of RPA32, we hypothesize that RPA32 phosphorylation is a crucial step within the DNA damage response. In other studies, we show that the radiosensitizing agent caffeine inhibits substrate phosphorylation by DNA-PK in vitro and in vivo. We also demonstrate that DNA-PK autophosphorylation at a cluster of residues plays an important role in DSB repair by regulating access to DNA ends. Lastly, we provide evidence that autophosphorylation of DNA-PK within the kinase domain of its catalytic subunit may cause inactivation of the protein kinase. Thus the focus of this thesis is to elucidate the role of RPA phosphorylation and DNA-PK-mediated protein phosphorylation in the DNA damage response. |
