DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract.
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.
Singhal
R K
RK
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-1068.
Prasad
R
R
Wilson
S H
SH
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism*; D004260:DNA Repair; D008297:Male; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013737:Testis / Q000378:metabolism*; D014498:Uracil / Q000378:metabolism*
D014498:Uracil; D004247:DNA; D004256:DNA Polymerase I