DNA-POLYMERASE-BETA CONDUCTS THE GAP-FILLING STEP IN URACIL-INITIATED BASE EXCISION-REPAIR IN A BOVINE TESTIS NUCLEAR EXTRACT
DNA-POLYMERASE-BETA CONDUCTS THE GAP-FILLING STEP IN URACIL-INITIATED BASE EXCISION-REPAIR IN A BOVINE TESTIS NUCLEAR EXTRACT
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DOI：
10.1074/jbc.270.2.949
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发表时间：
1995-01-13
影响因子：
4.8
通讯作者：
WILSON, SH
中科院分区：
文献类型：
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作者：
SINGHAL, RK;PRASAD, R;WILSON, SH
基因组DNA中GU错配主要由胞嘧啶残基脱氨基引起，通过碱基切除修复途径进行修复。使用含有单个G：U错配的51个碱基对的合成DNA底物，并且在修复期间掺入dCMP专门用于替换尿嘧啶。针对DNA聚合酶β（beta-pol）的中和多克隆抗体抑制修复反应，ddCTP也抑制修复反应，而aphidicolin没有显著影响，表明需要beta-pol的活性。接下来，使用部分纯化的组分重构碱基切除修复系统。解决了所需的几种酶活性，使得DNA连接酶和尿嘧啶-DNA糖基化酶/脱嘌呤/脱嘧啶核酸内切酶活性与DNA聚合酶要求分离。我们发现，纯化的β-pol可以恢复完整的DNA修复活性的DNA聚合酶耗尽的馏分，而纯化的DNA聚合酶α，δ，和β-pol不能，这些结果与纯化的蛋白质证实了与粗提物获得的结果，并表明β-pol是负责在这个体外碱基切除修复系统中涉及的单核苷酸缺口填充反应。
The GU 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.