CHARACTERIZATION OF THE DNA-POLYMERASE REQUIREMENT OF HUMAN BASE EXCISION-REPAIR

Base excision repair is one of the major mechanisms by which cells cor rect damaged DNA, We have developed an in vitro assay for base excisio n repair which is dependent on a uracil-containing DNA template, In th is report, we demonstrate the fractionation of a human cell extract in to two required components, One fraction was extensively purified and by several criteria shown to be identical to DNA polymerase beta (Pol beta). Purified, recombinant Pol beta efficiently substituted for this fraction, Escherichia coil Poll, mammalian Pol delta and to a lesser extent Pol alpha and epsilon also functioned in this assay. We provide evidence that multiple polymerases function in base excision repair i n human cell extracts. A neutralizing antibody to Pol beta, which inhi bited repair synthesis catalyzed by pure Pol beta by similar to 90%, o nly suppressed repair in crude extracts by a maximum of similar to 70% , An inhibitor of Pol beta, ddCTP, decreased base excision repair in c rude extracts by similar to 50%, whereas the Pol alpha/delta/epsilon i nhibitor, aphidicolin, reduced the reaction by similar to 20%, A combi nation of these chemical inhibitors almost completely abolished repair synthesis. These data suggest that Pol beta is the major base excisio n repair polymerase in human cells, but that other polymerases also co ntribute to a significant extent.