DNA Polymerase β Conducts the Gap-filling Step in Uracil-initiated Base Excision Repair in a Bovine Testis Nuclear Extract

Abstract

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 β (β-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of β-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 β-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases α, δ , and ε could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that β-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system. 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 β (β-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of β-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 β-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases α, δ , and ε could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that β-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.