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 fraction ate and purify proteins from a human cell extract that are involved in this type of repair. Three fractions are required to reconstitute bas e excision repair synthesis using a uracil-containing DNA as a model s ubstrate, We previously showed that one fraction corresponds to DNA po lymerase beta. A second fraction was extensively purified and found to possess uracil-DNA glycosylase activity and was identified as the pro duct 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 th e major polypeptide in fractions possessing DNA repair activity. Recom binant versions of UNG, HAP1, and DNA polymerase beta were able to sub stitute for the proteins purified from human cells. Addition of DNA li gase I led to ligated repair products. Thus, complete base excision re pair of uracil-containing DNA was achieved by a combination of UNG, HA P1, DNA polymerase beta, and DNA ligase I, This is the first complete reconstitution of base excision repair using entirely eukaryotic prote ins.