PROLIFERATING CELL NUCLEAR ANTIGEN-DEPENDENT ABASIC SITE REPAIR IN XENOPUS-LAEVIS OOCYTES - AN ALTERNATIVE PATHWAY OF BASE EXCISION DNA-REPAIR

DNA damage frequently leads to the production of apurinic/apyrimidinic (AP) sites, which are presumed to be repaired through the base excisi on pathway. For detailed analyses of this repair mechanism, a syntheti c analog of an AP site, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetr ahydrofuran), has been employed in a model system. Tetrahydrofuran res idues are efficiently repaired in a Xenopus laevis oocyte extract in w hich most repair events involve ATP-dependent incorporation of no more than four nucleotides (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989; Y. Matsumoto and D. F. Bogenhagen, Mel. Cell. Biol. 11:4441-4447, 1991). Using a series of column chromatography pr ocedures to fractionate X. laevis ovarian extracts, we developed a rec onstituted system of tetrahydrofuran repair with five fractions, three of which were purified to near homogeneity: proliferating cell nuclea r antigen (PCNA), AP endonuclease, and DNA polymerase delta. This PCNA -dependent system repaired natural AP sites as well as tetrahydrofuran residues. DNA polymerase beta was able to replace DNA polymerase delt a only for repair of natural AP sites in a reaction that did not requi re PCNA. DNA polymerase alpha did not support repair of either type of AP site. This result indicates that AP sites can be repaired by two d istinct pathways, the PCNA-dependent pathway and the DNA polymerase be ta-dependent pathway.