RECONSTITUTION OF HUMAN BASE EXCISION REPAIR WITH PURIFIED PROTEINS

Base excision repair is a major mechanism for correcting aberrant DNA bases. We are using an in vitro base excision repair assay to fractionate and purify proteins from a human cell extract that are involved in this type of repair. Three fractions are required to reconstitute base excision repair synthesis using a uracil-containing DNA as a model substrate. We previously showed that one fraction corresponds to DNA polymerase β. A second fraction was extensively purified and found to possess uracil-DNA glycosylase activity and was identified as the product of the UNG gene. A neutralizing antibody to the human UNG protein inhibited base excision repair in crude extract by at least 90%. The third fraction was highly purified and exhibited apurinic/apyrimidinic (AP) endonuclease activity. Immunoblot analysis identified HAP1 as the major polypeptide in fractions possessing DNA repair activity. Recombinant versions of UNG, HAP1, and DNA polymerase β were able to substitute for the proteins purified from human c...