Identification of 5′-deoxyribose phosphate lyase activity in human DNA polymerase γ and its role in mitochondrial base excision repair in vitro
Identification of 5′-deoxyribose phosphate lyase activity in human DNA polymerase γ and its role in mitochondrial base excision repair in vitro
复制标题
DOI：
10.1073/pnas.95.21.12244
复制
发表时间：
1998-10-13
影响因子：
11.1
通讯作者：
Copeland, WC
中科院分区：
文献类型：
--
作者：
Longley, MJ;Prasad, R;Copeland, WC
已经提出线粒体具有碱基切除修复过程以纠正线粒体基因组的氧化损伤。作为唯一的。DNA聚合酶（pol）存在于线粒体中，pol γ必然参与这些过程。因此，我们测试了人聚合酶γ的催化亚基参与尿嘧啶引起的碱基切除修复的能力，用纯化的组分在体外重建。在尿嘧啶-DNA糖基化酶和脱嘌呤/脱嘧啶核酸内切酶的作用之后，人pol γ能够在5'末端脱氧核糖磷酸（dRP）瓣存在下填充单核苷酸缺口。我们在这里报告，人聚γ的催化亚基催化释放的dRP残基从切割脱嘌呤/脱嘧啶位点产生的DNA连接酶的底物。这种活性的热敏性表明dRP裂解酶功能需要三维蛋白质结构。dRP裂解酶活性不需要二价金属离子，并且用NaBH 4捕获共价酶-DNA复合物的能力强烈暗示了β-消除反应机制中的席夫碱中间体。
Mitochondria have been proposed to possess base excision repair processes to correct oxidative damage to the mitochondrial genome. As the only. DNA polymerase (pol) present in mitochondria, pol gamma is necessarily implicated in such processes. Therefore, we tested the ability of the catalytic subunit of human pol gamma to participate in uracil-provoked base excision repair reconstituted in vitro with purified components. Subsequent to actions of uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease, human pol gamma was able to fill a single nucleotide gap in the presence of a 5' terminal deoxyribose phosphate (dRP) flap. We report here that the catalytic subunit of human pol gamma catalyzes release of the dRP residue from incised apurinic/apyrimidinic sites to produce a substrate for DNA ligase. The heat sensitivity of this activity suggests the dRP lyase function requires a three-dimensional protein structure. The dRP lyase activity does not require divalent metal ions, and the ability to trap covalent enzyme-DNA complexes with NaBH4 strongly implicates a Schiff base intermediate in a beta-elimination reaction mechanism.