Mammalian base excision repair by DNA polymerases delta and epsilon.
Two distinct pathways for completion of base excision repair (BER) have been discovered in eukaryotes: the DNA polymerase beta (Pol beta)-dependent 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 either Pol delta or Pol epsilon. These fractions were then tested for their ability to perform both short- and long-patch BER in an in vitro repair assay, using a circular DNA template, containing a single abasic site at a defined position. Remarkably, both Pol delta and Pol epsilon were able to replace a single nucleotide at the lesion site, but the 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 the first time that Pol delta and/or Pol epsilon are directly involved in the long-patch BER of abasic sites and might function as back-up system for Pol beta in one-gap filling reactions.
Stucki
M
M
Institut für Veterinärbiochemie, Universität Zürich, Switzerland.
Pascucci
B
B
Parlanti
E
E
Fortini
P
P
Wilson
S H
SH
Hübscher
U
U
Dogliotti
E
E
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004257:DNA Polymerase II / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000172:deficiency / Q000235:genetics; D004260:DNA Repair; D005347:Fibroblasts; D017353:Gene Deletion; D007700:Kinetics; D008322:Mammals; D051379:Mice; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D013379:Substrate Specificity
D018809:Proliferating Cell Nuclear Antigen; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D019951:DNA Polymerase beta