AP Endonucleases and the Many Functions of Ref-1 (2001)

APE-1/Ref-1 is a ubiquitous multifunctional protein that possesses both DNA repair activity and redox regulatory activity. Originally named apurinic/apyrimidinic endonuclease (APE-1) and HAP-1 (Human APE-1) for its endonuclease activity, APE-1/Ref-1 is a major enzyme in the base excision repair pathway (1) and is involved in repair of spontaneous and oxidative damage to cellular DNA (2). In signal transduction, APE-1/Ref-1 is important in mediating DNA binding of a number of transcription factors including AP-1, nuclear factor (NF)B, Pax-5, Pax-8, HIF-1, and HLF (Table 1) (2–8). Although the nomenclature is not standardized, many authors refer to the protein as APE-1/ Ref-1 to reflect its dual function. APE-1/Ref-1 is ubiquitously expressed, though there are complex patterns of distribution within cells and between different cell types (9–13). These patterns of cellular distribution may be related to the varying functions of APE-1/Ref-1. Based on its multiple functions, APE-1/Ref-1 likely plays an important role in maintaining genomic integrity and in regulating gene expression via redox activation of a variety of transcription factors. Human cells in vitro are estimated to undergo spontaneous depurination of DNA at a rate of approximately 10,000 bases/day/cell (14), with the greatest number of apurinic/apyrimidinic (AP) sites occurring in the brain, heart, and colon (15). In addition to spontaneous loss of nucleotides, DNA can also be damaged via oxidant stress that results in oxidation of bases and sugars (16). To protect the integrity of the genome, all cells have developed a repair system to excise and replace the damaged nucleotides (AP sites) in DNA. In the base excision repair pathway, damaged bases are excised by a DNA glycosylase, creating an AP site. APE-1/Ref-1 cleaves the DNA backbone 5 to the AP site and repair is completed by DNA polymerase and DNA ligase (17–19). There are two families of endonucleases that are differentiated based on their functions and homology to Escherichia coli exonucleases (20). APE-1/Ref-1 belongs to the first of two families of AP endonucleases and shares sequence homology with E. coli exonuclease III. It accounts for approximately 95% of DNA repair capabilities in humans. In the yeast Saccharomyces cerevisiae , AP endonuclease-1 (APN-1) is the primary AP endonuclease and accounts for the majority of the yeast DNA repair activity. In contrast to APE-1/Ref-1, APN-1 belongs to the second family of AP endonucleases and shares homology with E. coli exonuclease IV (21). Though both enzymes have comparable 5 endonuclease activity and the ability to repair AP sites, APN-1 has much greater 3 -diesterase activity, and no known redox capability. In APE-1/Ref-1, the DNA repair activity resides in the C-terminal portion of the protein, while the N-terminal domain is necessary for redox regulation of transcription (22). In addition to the 90% homology between various mammalian AP endonucleases, there is a highly conserved cysteine at position 65 in human, mouse, and rat AP endonucleases that is thought to be important in redox function of these proteins. APE-1/Ref-1 functions as a regulatory protein for redox activation of a number of transcription factors (2–8). Xanthoudakis and Curran (2) originally identified the redox function of APE-1/Ref-1 when they studied the in vitro activation of AP-1 DNA binding in HeLa cells. APE-1/ Ref-1 acts via a post-translational mechanism in which conserved cysteine residues in the DNA-binding domains of Fos and Jun proteins are reduced, allowing DNA binding to occur. We have demonstrated a direct correlation between the amounts of nuclear APE-1/Ref-1 and AP-1 activity in alveolar macrophages, suggesting an important role for APE-1/Ref-1 in the inflammatory response in the lung (23). AP-1 is one of many transcription factors under redox control of APE-1/Ref-1. Table 1 lists the transcription factors shown to exhibit redox regulation by APE-1/ Ref-1. For example, Jayaraman and colleagues (24) demonstrated that cotransfection of p53, a transcription factor that is involved in the response to oxidative stress and apoptosis, with APE-1/Ref-1, increases expression of the p53-dependent genes, cyclin G, p21, and BAX. Additionally, the Pax family of genes, important for normal development, cellular differentiation, and thyroid function, has been shown to exhibit transcriptional activation of specific promotors in cotransfection experiments with APE-1/Ref-1 (7, 8). These and other transcription factors under redox control of APE-1/Ref-1 are involved in regulation of a number of important cellular functions and these studies suggest a complex, multifunctional role for APE-1/Ref-1 in cellular response to stress. Regulation of APE-1/Ref-1 occurs at both the transcriptional and post-translational level. Transcriptional regulation of APE-1/Ref-1 has been shown to occur via a ( Received in original form September 22, 2001 )