BACKGROUND Mutations in human gene encoding the mitochondrial DNA polymerase γ (HsPolγ) are associated with a broad range of mitochondrial diseases. Here we studied the impact on DNA replication by disease variants clustered around residue HsPolγ-K1191, a residue that in several family-A DNA polymerases interacts with the 3' end of the primer. METHODS Specifically, we examined the effect of HsPolγ carrying pathogenic variants in residues D1184, I1185, C1188, K1191, D1196, and a stop codon at residue T1199, using as a model the yeast mitochondrial DNA polymerase protein, Mip1p. RESULTS The introduction of pathogenic variants C1188R (yV945R), and of a stop codon at residue T1199 (yT956X) abolished both polymerization and exonucleolysis in vitro. HsPolγ substitutions in residues D1184 (yD941), I1185 (yI942), K1191 (yK948) and D1196 (yD953) shifted the balance between polymerization and exonucleolysis in favor of exonucleolysis. HsPolγ pathogenic variants at residue K1191 (yK948) and D1184 (yD941) were capable of nucleotide incorporation albeit with reduced processivity. Structural analysis of mitochondrial DNAPs showed that residue HsPolγ-N864 is placed in an optimal distance to interact with the 3' end of the primer and the phosphate backbone previous to the 3' end. Amino acid changes in residue HsPolγ-N864 to Ala, Ser or Asp result in enzymes that did not decrease their polymerization activity on short templates but exhibited a substantial decrease for processive DNA synthesis. CONCLUSION Our data suggest that in mitochondrial DNA polymerases multiple amino acids are involved in the primer-stand stabilization.

中文翻译：

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线粒体 DNA 聚合酶致病变异的建模：深入了解复制缺陷和对人类疾病的影响。

背景编码线粒体DNA聚合酶γ(HsPolγ)的人类基因的突变与广泛的线粒体疾病有关。在这里，我们研究了聚集在残基 HsPolγ-K1191 周围的疾病变异对 DNA 复制的影响，HsPolγ-K1191 是几个家族 A DNA 聚合酶中与引物 3' 端相互作用的残基。方法 具体而言，我们使用酵母线粒体 DNA 聚合酶蛋白 Mip1p 作为模型，检测了携带致病变异的 HsPolγ 在残基 D1184、I1185、C1188、K1191、D1196 和残基 T1199 处的终止密码子中的影响。结果 致病变异 C1188R (yV945R) 和 T1199 残基处的终止密码子 (yT956X) 的引入消除了体外聚合和核酸外切。残基 D1184 (yD941)、I1185 (yI942)、K1191 (yK948) 和 D1196 (yD953) 改变了聚合和核酸外切之间的平衡，有利于核酸外切。残基 K1191 (yK948) 和 D1184 (yD941) 处的 HsPolγ 致病变异能够掺入核苷酸，尽管持续合成能力降低。线粒体 DNAP 的结构分析表明，残基 HsPolγ-N864 放置在最佳距离内，以便与引物的 3' 端和 3' 端之前的磷酸骨架相互作用。残基 HsPolγ-N864 中的氨基酸变为 Ala、Ser 或 Asp 导致酶不会降低其对短模板的聚合活性，但会显着降低持续性 DNA 合成。结论 我们的数据表明，在线粒体 DNA 聚合酶中，多种氨基酸参与了引物-链的稳定性。