DNA synthesis and dRPase activities of polymerase beta are both essential for single-nucleotide patch base excision repair in mammalian cell extracts.
In mammalian cells the majority of altered bases in DNA are processed through a single-nucleotide patch base excision repair mechanism. Base excision repair is initiated by a DNA glycosylase that removes a damaged base and generates an abasic site (AP site). This AP site is further processed by an AP endonuclease activity that incises the phosphodiester bond adjacent to the AP site and generates a strand break containing 3'-OH and 5'-sugar phosphate ends. In mammalian cells, the 5'-sugar phosphate is removed by the AP lyase activity of DNA polymerase beta (Pol beta). The same enzyme also fills the gap, and the DNA ends are finally rejoined by DNA ligase. We measured repair of oligonucleotide substrates containing a single AP site in cell extracts prepared from normal and Pol beta-null mouse cells and show that the reduced repair in Pol beta-null extracts can be complemented by addition of purified Pol beta. Using this complementation assay, we demonstrate that mutated Pol beta without dRPase activity is able to stimulate long patch BER. Mutant Pol beta deficient in DNA synthesis, but with normal dRPase activity, does not stimulate repair in Pol beta-null cells. However, under conditions where we measure base excision repair accomplished exclusively through a single-nucleotide patch BER, neither dRPase nor DNA synthesis mutants of Pol beta alone, or the two together, were able to complement the repair defect. These data suggest that the dRPase and DNA synthesis activities of Pol beta are coupled and that both of these Pol beta functions are essential during short patch BER and cannot be efficiently substituted by other cellular enzymes.
Podlutsky
A J
AJ
Laboratory of Molecular Genetics, National Institute on Aging, NIH, Baltimore, Maryland 21224, USA.
Dianova
I I
II
Wilson
S H
SH
Bohr
V A
VA
Dianov
G L
GL
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002474:Cell-Free System / Q000201:enzymology*; D019951:DNA Polymerase beta / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000172:deficiency / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology; D005816:Genetic Complementation Test; D051379:Mice; D018345:Mice, Knockout; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D014498:Uracil / Q000378:metabolism
D009841:Oligonucleotides; D014498:Uracil; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase; C058246:DNA deoxyribophosphodiesterase; D010727:Phosphoric Diester Hydrolases