DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract

J Biol Chem. 1995 Jan 13;270(2):949-57. doi: 10.1074/jbc.270.2.949.


The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway. We found that a bovine testis crude nuclear extract conducts uracil-initiated base excision repair in vitro. A 51-base pair synthetic DNA substrate containing a single G:U mismatch was used, and incorporation of dCMP during repair was exclusively to replace uracil. A neutralizing polyclonal antibody against DNA polymerase beta (beta-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of beta-pol was required. Next, the base excision repair system was reconstituted using partially purified components. Several of the enzymatic activities required were resolved, such that DNA ligase and the uracil-DNA glycosylase/apurinic/apyrimidinic endonuclease activities were separated from the DNA polymerase requirement. We found that purified beta-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases alpha, delta, and epsilon could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that beta-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.

MeSH terms

  • Animals
  • Base Sequence
  • Cattle
  • Cell Nucleus / metabolism*
  • Chromatography, Ion Exchange
  • DNA / genetics
  • DNA / metabolism
  • DNA Polymerase I / metabolism*
  • DNA Repair*
  • Male
  • Molecular Sequence Data
  • Saccharomyces cerevisiae / genetics
  • Testis / metabolism*
  • Uracil / metabolism*


  • Uracil
  • DNA
  • DNA Polymerase I