Singhal R K, Prasad R, Wilson S H
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-1068.
J Biol Chem. 1995 Jan 13;270(2):949-57. doi: 10.1074/jbc.270.2.949.
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.
基因组DNA中的G:U错配主要源于胞嘧啶残基的脱氨基作用，并通过碱基切除修复途径进行修复。我们发现牛睾丸粗核提取物在体外能进行尿嘧啶引发的碱基切除修复。使用了一个含有单个G:U错配的51个碱基对的合成DNA底物，修复过程中dCMP的掺入专门用于取代尿嘧啶。一种针对DNA聚合酶β（β-pol）的中和多克隆抗体抑制了修复反应。ddCTP也抑制了修复反应，而阿非科林没有显著影响，这表明需要β-pol的活性。接下来，使用部分纯化的组分重建碱基切除修复系统。解析出了所需的几种酶活性，从而将DNA连接酶和尿嘧啶-DNA糖基化酶/无嘌呤/无嘧啶内切核酸酶活性与对DNA聚合酶的需求分离开来。我们发现纯化的β-pol可以将完全的DNA修复活性恢复到缺乏DNA聚合酶的组分中，而纯化的DNA聚合酶α、δ和ε则不能。这些用纯化蛋白得到的结果证实了用粗提取物获得的结果，并表明β-pol负责该体外碱基切除修复系统中涉及的单核苷酸缺口填补反应。
