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首页> 外文期刊>Biochemistry >Characterization of the native and recombinant catalytic subunit of human DNA polymerase gamma: Identification of residues critical for exonuclease activity and dideoxynucleotide sensitivity
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Characterization of the native and recombinant catalytic subunit of human DNA polymerase gamma: Identification of residues critical for exonuclease activity and dideoxynucleotide sensitivity

机译：人类DNA聚合酶γ的天然和重组催化亚基的表征：鉴定对核酸外切酶活性和双脱氧核苷酸敏感性至关重要的残基

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摘要

The human DNA polymerase gamma catalytic subunit was overexpressed in recombinant baculovirus-infected insect cells, and the 136 000 Da protein was purified to homogeneity. Application of the same purification protocol to HeLa mitochondrial lysates permitted isolation of native DNA polymerase gamma as a single subunit, allowing direct comparison of the native and recombinant enzymes without interference of other polypeptides. Both forms exhibited identical properties, and the DNA polymerase and 3' --> 5' exonuclease activities were shown unambiguously to reside in the catalytic polypeptide. The salt sensitivity and moderate processivity of the isolated catalytic subunit suggest other factors could be required to restore the salt tolerance and highly processive DNA synthesis typical of gamma polymerases. To facilitate our understanding of mitochondrial DNA replication and mutagenesis as well as cytotoxicity mediated by antiviral nucleotide analogues, we also constructed two site-directed mutant proteins of the human DNA polymerase gamma. Substituting alanine for two essential acidic residues in the exonuclease motif selectively eliminated the 3' --> 5' exonucleolytic function of the purified mutant polymerase gamma. Replacement of a tyrosine residue critical for sugar recognition with phenylalanine in polymerase motif B reduced dideoxynucleotide inhibition by a factor of 5000 with only minor effects on overall polymerase function. [References: 48]
机译：人DNA聚合酶γ催化亚基在重组杆状病毒感染的昆虫细胞中过表达，并且将136 000 Da蛋白纯化至同质。将相同的纯化方案应用于HeLa线粒体裂解物，可以分离天然DNA聚合酶γ作为单个亚基，从而可以直接比较天然酶和重组酶，而不会干扰其他多肽。两种形式均表现出相同的性质，并且DNA聚合酶和3'-> 5'核酸外切酶活性明确显示存在于催化多肽中。分离的催化亚基的盐敏感性和适度的合成能力表明，可能需要其他因素来恢复典型的伽玛聚合酶的耐盐性和高合成DNA合成能力。为了促进我们对线粒体DNA复制和诱变以及抗病毒核苷酸类似物介导的细胞毒性的了解，我们还构建了人类DNA聚合酶gamma的两个定点突变蛋白。用丙氨酸替代核酸外切酶基序中的两个必需的酸性残基，可选择性消除纯化的突变聚合酶γ的3'-> 5'核酸外切功能。在聚合酶基序B中用苯丙氨酸替代对糖识别至关重要的酪氨酸残基，可将双脱氧核苷酸抑制作用降低5000倍，而对整体聚合酶功能的影响却很小。 [参考：48]

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