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 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.
Highlights
Matsumoto and Bogenhagen (0) argued that,B-pol may be responsible for gap filling during repair of a tetrahydrofuran lesion by BER in a Xenopus Iaeuis oocyte extract, and more recently Dianov et al [11], using a human cell nuclear extract system for uracil-initiated BER, obtained inhibition by dideoxynucleotide; this led these workers to conclude that,B-pol was responsible for the DNA synthesis step
The polymerase requirement in the lymphoblastoid cell line-based BER system studied by Dianov et al [11] was not clear cut, First, the reaction was inhibited by the a-pol inhibitor aphidicolin and only partially blocked by the,B-pol inhibitor ddNTP, yet purified mammalian,B-pol is not inhibited by 100 !kg/ml aphidicolin and is >95% inhibited by ddNTP at a ddNTP/dNTP ratio of only 10 [12, 13]
Our results indicate that DNA polymerase {3 is solely responsible for the single nucleotide gap-filling synthesis in uracil-initiated BER in bovine testis nuclear extracts but not in S. cereuisiae
Summary
(Received for publication, August 5, 1994, and in revised form, October 26, 1994). Rakesh K. We found that purified P-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases a, 0, and E could not These results with purified proteins corroborated results obtained with the crude extract and indicate that P-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system. [14] have suggested that results on yeast DNA polymerase requirements in DNA repair may be extrapolated to mammalian DNA repair In light of these ambiguities and apparent contradictions, and earlier demonstrations that purified {3-pol can completely fill short gaps in vitro [17,18,19], we undertook a study to further examine the putative role of {3-pol in uracil-initiated base excision repair. Our results indicate that DNA polymerase {3 is solely responsible for the single nucleotide gap-filling synthesis in uracil-initiated BER in bovine testis nuclear extracts but not in S. cereuisiae
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