Abstract
Base excision repair (BER) is one of the dominant pathways to repair altered DNA bases, and is well conserved from bacteria to mammals. Single nucleotide BER (SN-BER) is a simple pathway to remove a single nucleotide. We have investigated several protein–protein interactions and their coordination in SN-BER initiated by methylpurine-DNA glycosylase (MPG). MPG recognizes and excises damaged purines, such as hypoxantin or methylated bases, from DNA. With activity assays using radio-labeled duplex oligonucleotide substrates, we concluded that the downstream enzymes increase the upstream enzyme activity in SN-BER. For example, AP endonuclease 1 (APE1) increased MPG activity, and DNA polymerase β increased MPG and APE1 activity. Since the stimulations resulted from increases of kcat values, a downstream enzyme would accelerate the disassociation of the upstream enzyme from the product. Intracellular X-ray repair cross complementing protein 1 (XRCC1) is thought to make complexes with 80 percent of DNA Ligase 3 (LIG3) molecules under physiological conditions, and interacts with almost all of BER enzymes to stabilize the configuration. In the presence of recombinant XRCC1, kcat/Km value of MPG and APE1 activity increased 1.8-fold and 7.1-fold, respectively, suggesting the involvement in a repair complex during the early step of SN-BER pathway. The studies on the effects of XRCC1-LIG3 complex to MPG and APE1 activities are in progress.
