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• W2000479074 endingPage "957" @default.
• W2000479074 startingPage "949" @default.
• W2000479074 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." @default.
• W2000479074 created "2016-06-24" @default.
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• W2000479074 date "1995-01-01" @default.
• W2000479074 modified "2023-10-18" @default.
• W2000479074 title "DNA Polymerase β Conducts the Gap-filling Step in Uracil-initiated Base Excision Repair in a Bovine Testis Nuclear Extract" @default.
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• W2000479074 doi "https://doi.org/10.1074/jbc.270.2.949" @default.
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