Repair of oxidative DNA damage in mammalian cells

DNA base excision repair (BER) is a ubiquitous and indispensable defense mechanism against cell death, mutagenesis, and carcinogenesis caused by spontaneously formed DNA damage, such as oxidation and alkylation. Apurinic/apyrimidinic endonuclease (APE1 in mammals) arguably determines the BER rate in cells and thus plays a central role in the repair pathway. Recent findings indicate that APE1 is essential for cellular viability, and that endogenous DNA damage is lethal if left unrepaired. Independently from its repair activity, APE1 interacts with histone deacetylase 1 (HDAC1) to suppress expression of many genes, including the parathyroid hormone gene. APE1 also regulates cJun/cFos and other crucial transcription factors. A high expression of APE1 is often observed in malignant tumor cells, which are more resistant to radio- and chemotherapeutic treatments, suggesting that APE1’s repair function increases the chance of survival for cancer cells as well. Thus, APE1 may be either beneficial or harmful to humans, depending on the type of cells in which it is expressed. Understanding the mechanism for regulation of APE1 activity is therefore crucial for evaluating BER efficiency and its consequences in normal and cancer cells. Indeed, cancer predisposition by APE1 deficiency is known and p53 is an upstream regulator of APE1. We have discovered that APE1 is ubiquitinated by mouse double minute 2 (MDM2), and that the ubiquitination is induced by genotoxic stress in a p53-dependent manner. Ubiquitination occurs at specific Lys residues in the APE1 polypeptide, and appears to have significant effects on the functions of APE1 and other cellular proteins, including XRCC1 (X-ray cross-complementation 1), p53, and HDAC1, that interact directly with APE1. The central hypothesis of this project is that APE1 ubiquitination, regulated by MDM2 and p53, modulates the APE1 level and functions in DNA damage response. In order to elucidate the cellular defense mechanism involving this remarkably multifunctional protein, we will: (1) determine the effect of APE1 ubiquitination on its enzymatic and DNA binding activities; (2) unravel interaction of ubiquitinated APE1 with factors in BER and p53 signaling network; and (3) establish the regulation of APE1 ubiquitination in vivo. These parameters are crucial to predict susceptibility of cancer cells against therapeutic drugs that generates genomic instability. With the research facilities provided by Louisiana Cancer Research Consortium (LCRC), this project in the long-term should provide insights for developing new modalities for adjuvant cancer therapies and organ preservation, and for improving the management of other age-related pathophysiology.