Antimutator Alleles of Yeast DNA Polymerase Gamma Modulate the Balance between DNA Synthesis and Excision
Antimutator Alleles of Yeast DNA Polymerase Gamma Modulate the Balance between DNA Synthesis and Excision
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DOI：
10.1371/journal.pone.0027847
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发表时间：
2011-11-16
期刊：
影响因子：
3.7
通讯作者：
Szczepanowska, Karolina
中科院分区：
文献类型：
--
作者：
Foury, Francoise;Szczepanowska, Karolina
线粒体DNA （mtDNA）突变是人类疾病甚至衰老的重要原因。DNA聚合酶γ (pol γ)是线粒体内独特的复制酶，通过选择正确的核苷酸和3′-5′核酸外切酶的校对，对mtDNA复制的保真度起着关键作用。我们首次分离并鉴定了酵母pol γ (Mip1)的抗突变等位基因。这些mip1突变位于3‘-5’核酸外切酶和聚合酶结构域，在mtDNA错配修复部分缺陷的msh1-1菌株中引起mtDNA点突变频率降低2-15倍。体外实验表明，与野生型相比，在所有突变体中，DNA合成和外核溶解之间的平衡都向切除转移，这表明在体内，编辑功能有更多的机会消除复制错误。结果部分补偿了错配修复缺陷，降低了mtDNA点突变率。然而，在野生型MSH1背景下，除一个突变体外，所有突变体都失去了抗突变性状。因此，所选突变体的聚合酶表现出较少的寡核苷酸引物M13 ssDNA合成和较小程度的DNA结合亲和力，这表明在错配修复熟练的细胞中，需要高效的DNA合成才能达到最佳精度。相比之下，Mip1-A256T聚合酶表现出野生型DNA合成活性，在MSH1和MSH1 -1背景下都能提高mtDNA复制保真度。总之，我们的数据表明，野生型Mip1的准确性可能不是最佳的，可以通过特定的（通常是保守的）氨基酸替换来提高，这些替换定义了一个pol γ区域，包括棕榈亚结构域的环，ExoII基元附近的两个残基以及靠近聚合酶结构域的外切酶螺旋-线圈-螺旋模块。这些元素以一种微妙的方式调节着DNA聚合和DNA切除之间的平衡。
Mutations in mitochondrial DNA (mtDNA) are an important cause of disease and perhaps aging in human. DNA polymerase gamma (pol gamma), the unique replicase inside mitochondria, plays a key role in the fidelity of mtDNA replication through selection of the correct nucleotide and 3'-5' exonuclease proofreading. For the first time, we have isolated and characterized antimutator alleles in the yeast pol gamma (Mip1). These mip1 mutations, localised in the 3'-5' exonuclease and polymerase domains, elicit a 2-15 fold decrease in the frequency of mtDNA point mutations in an msh1-1 strain which is partially deficient in mtDNA mismatch-repair. In vitro experiments show that in all mutants the balance between DNA synthesis and exonucleolysis is shifted towards excision when compared to wild-type, suggesting that in vivo more opportunity is given to the editing function for removing the replicative errors. This results in partial compensation for the mismatch-repair defects and a decrease in mtDNA point mutation rate. However, in all mutants but one the antimutator trait is lost in the wild-type MSH1 background. Accordingly, the polymerases of selected mutants show reduced oligonucleotide primed M13 ssDNA synthesis and to a lesser extent DNA binding affinity, suggesting that in mismatch-repair proficient cells efficient DNA synthesis is required to reach optimal accuracy. In contrast, the Mip1-A256T polymerase, which displays wild-type like DNA synthesis activity, increases mtDNA replication fidelity in both MSH1 and msh1-1 backgrounds. Altogether, our data show that accuracy of wild-type Mip1 is probably not optimal and can be improved by specific (often conservative) amino acid substitutions that define a pol gamma area including a loop of the palm subdomain, two residues near the ExoII motif and an exonuclease helix-coil-helix module in close vicinity to the polymerase domain. These elements modulate in a subtle manner the balance between DNA polymerization and excision.