DNA single-strand breaks (SSBs), generated by reactive oxygen species (ROS) directly or indirectly, activate poly (ADP-ribose) polymerase (PARP) whose role in the DNA base excision repair (BER) is implicated. However, if overactivated, PARP can cause necrosis and inflammation in many pathophysiological conditions such as ischemia/reperfusion. Our studies indicate that the mammalian apurinic/apyrimidinic endonuclease (APE1), an essential BER protein, forms a stable complex with SSBs, and thus protects the lesions from being recognized by PARP. We have also found that APE1 can be acetylated by the histone acetyltransferase p300, which decreases APE1's affinity for the cleaved DNA. The central hypothesis of this project is that APE1's ability to bind to SSBs is crucial for cellular recovery from ROS-generated DNA damage, and for protection of cells from necrosis caused by PARP overactivation. Our hypothesis emphasizes the role of non-enzymatic APE1 functions, and will be tested by investigating (1) how APE1 competes with PARP for SSB binding; (2) how the acetylation modulates APE1 activity; and (3) how APE1 suppresses PARP overactivation in vitro and in vivo. We will determine the binding patterns and association constants of APE1 and PARP for various types of SSBs by electrophoretic mobility and DNA footprinting assays, and by fluorescence anisotropy measurement. Various site-specific APE1 mutants, including polymorphic alleles, will be examined to identify and elucidate the role of amino acid side chains critical for SSB binding, which our preliminary results imply is modulated independently of catalysis. The DNA binding domain of PARP will also be separately characterized for its binding mechanism. We will examine how nucleosomal structure affects the accessibility of these proteins to SSBs. We will perform cell biological studies to understand the effects of the APE1 level on PARP activation and the cytotoxicity induced by ROS and other damage-inducing reagents. These studies will help us understand the cellular defense mechanism against ROS more precisely, and will advance our long-term goal: to correlate the cellular BER capacity to cancer risk and age-related pathophysiology, and to seek better ways for their prevention.
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