MAMMALIAN BASE EXCISION-REPAIR BY DNA-POLYMERASE-DELTA AND DNA-POLYMERASE-EPSILON

Two distinct pathways for completion of base excision repair (BER) hav e been discovered in eukaryotes: the DNA polymerase beta (Pol beta)-de pendent short-patch pathway that involves the replacement of a single nucleotide and the long-patch pathway that entails the resynthesis of 2-6 nucleotides and requires PCNA, We have used cell extracts from Pol beta-deleted mouse fibroblasts to separate subfractions containing ei ther Pol delta or Pol epsilon. These fractions were then tested for th eir ability to perform both short- and long-patch BER in an ill vitro repair assay, using a circular DNA template, containing a single abasi c site at a defined position. Remarkably, both Pol delta and Pol epsil on were able to replace a single nucleotide at the lesion site, but th e repair reaction is delayed compared to single nucleotide replacement by Pol beta, Furthermore, our observations indicated, that either Pol delta and/or Pol epsilon participate in the long-patch BER. PCNA and RF-C, but not RP-A are required for this process. Our data show for th e first time that Pol delta and/or Pol epsilon are directly involved i n the long-patch BER of abasic sites and might function as back-up sys tem for Pol beta in one-gap filling reactions.