Functions of the major abasic endonuclease (APE1) in cell viability and genotoxin resistance

Abstract

DNA is susceptible to a range of chemical modifications, with one of the most frequent lesions being apurinic/apyrimidinic (AP) sites. AP sites arise due to damage-induced (e.g. alkylation) or spontaneous hydrolysis of the N-glycosidic bond that links the base to the sugar moiety of the phosphodiester backbone, or through the enzymatic activity of DNA glycosylases, which release inappropriate bases as part of the base excision repair (BER) response. Unrepaired AP sites, which lack instructional information, have the potential to cause mutagenesis or to arrest progressing DNA or RNA polymerases, potentially causing outcomes such as cellular transformation, senescence or death. The predominant enzyme in humans responsible for repairing AP lesions is AP endonuclease 1 (APE1). Besides being a powerful AP endonuclease, APE1 possesses additional DNA repair activities, such as 3′-5′ exonuclease, 3′-phophodiesterase and nucleotide incision repair. In addition, APE1 has been shown to stimulate the DNA-binding activity of a number of transcription factors through its 'REF1' function, thereby regulating gene expression. In this article, we review the structural and biochemical features of this multifunctional protein, while reporting on new structures of the APE1 variants Cys65Ala and Lys98Ala. Using a functional complementation approach, we also describe the importance of the repair and REF1 activities in promoting cell survival, including the proposed passing-the-baton coordination in BER. Finally, results are presented indicating a critical role for APE1 nuclease activities in resistance to the genotoxins methyl methanesulphonate and bleomycin, supporting biologically important functions as an AP endonuclease and 3′-phosphodiesterase, respectively. Genotoxic stress can arise from altered cellular metabolism or following exposure to many external agents, including sunlight, ionising radiation, polycyclic aromatic hydrocarbons (e.g. benzo[a]pyrene), aromatic amines (e.g. 2-acetylaminofluorene), aflatoxins, various heavy metals (e.g. cadmium) and numerous therapeutic agents (e.g. X-rays, platins, alkylators, crosslinkers, topoisomerase poisons and antimetabolites). One of the more common forms of intracel-lular stress originates from an imbalance in reactive oxygen species (ROS) formation, typically stemming from defects in mitochondrial respiration or metabolism, and their sequestration by the protective scavenging systems. In conditions of so-called oxidative stress, the elevated ROS can attack nearby intracellular macromolecules, including proteins, lipids and nucleic acids, potentially giving rise to cellular dysfunction. As damage to one's genetic material can lead to permanent sequence changes or to alterations in DNA and/or RNA metabolism that can promote disease and ageing, DNA repair processes have evolved as a major front-line defense against the harmful consequences of genotoxic stress. In this article, we review the structural and biochemical features of this multifunctional protein, while reporting on new structures of the APE1 variants Cys65Ala and Lys98Ala. Using a functional complementation approach, we also describe the importance of the repair and REF1 activities in promoting cell survival, including the proposed passing-the-baton coordination in BER. Finally, results are presented indicating a critical role for APE1 nuclease activities in resistance to the genotoxins methyl methanesulphonate and bleomycin, supporting biologically important functions as an AP endonuclease and 3′-phosphodiesterase, respectively.