FEN1 stimulation of DNA polymerase beta mediates an excision step in mammalian long patch base excision repair.
In mammalian cells, single-base lesions, such as uracil and abasic sites, appear to be repaired by at least two base excision repair (BER) subpathways: "single-nucleotide BER" requiring DNA synthesis of just one nucleotide and "long patch BER" requiring multi-nucleotide DNA synthesis. In single-nucleotide BER, DNA polymerase beta (beta-pol) accounts for both gap filling DNA synthesis and removal of the 5'-deoxyribose phosphate (dRP) of the abasic site, whereas the involvement of various DNA polymerases in long patch BER is less well understood. Recently, we found that beta-pol plays a role in mammalian cell extract-mediated long patch BER, in that formation of a key excision product, 5'-dRP-trinucleotide (5'-dRP-N(3)), is dependent upon beta-pol (Dianov, G. L., Prasad, R., Wilson, S. H., and Bohr, V.A. (1999) J. Biol. Chem. 274, 13741-13743). The structure-specific endonuclease flap endonuclease 1 (FEN1) has also been suggested to be involved in long patch BER excision. Here, we demonstrate by immunodepletion experiments that 5'-dRP-N(3) excision in long patch BER of uracil-DNA in a human lymphoid cell extract is, indeed, dependent upon FEN1. Next, we reconstituted the excision step of long patch BER using purified human proteins and an oligonucleotide substrate with 5'-dRP at the margin of a one-nucleotide gap. Formation of the excision product 5'-dRP-N(3) was dependent upon both strand displacement DNA synthesis by beta-pol and FEN1 excision. FEN1 stimulated strand displacement DNA synthesis of beta-pol. FEN1 acting either alone, or without DNA synthesis by beta-pol, produced a two-nucleotide excision product, 5'-dRP-N(1), but not 5'-dRP-N(3). These results demonstrate that human FEN1 and beta-pol can cooperate in long patch BER excision and specify the predominant excision product seen with a cell extract.
Prasad
R
R
Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Dianov
G L
GL
Bohr
V A
VA
Wilson
S H
SH
D002460:Cell Line; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D019951:DNA Polymerase beta / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000494:pharmacology*; D045585:Flap Endonucleases; D006801:Humans; D009711:Nucleotides / Q000378:metabolism; D012267:Ribosemonophosphates / Q000378:metabolism
D009711:Nucleotides; D012267:Ribosemonophosphates; C058245:2-deoxyribose 5-phosphate; D004247:DNA; D019951:DNA Polymerase beta; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human; D043211:Exodeoxyribonuclease V