YEAST MITOCHONDRIAL-DNA MUTATORS WITH DEFICIENT PROOFREADING EXONUCLEOLYTIC ACTIVITY
YEAST MITOCHONDRIAL-DNA MUTATORS WITH DEFICIENT PROOFREADING EXONUCLEOLYTIC ACTIVITY
复制标题
DOI：
10.1002/j.1460-2075.1992.tb05337.x
复制
发表时间：
1992-07-01
期刊：
影响因子：
11.4
通讯作者：
VANDERSTRAETEN, S
中科院分区：
文献类型：
--
作者：
FOURY, F;VANDERSTRAETEN, S
编码酵母线粒体 DNA 聚合酶的 MIP1 基因在其 N 末端区域具有三个基序（Exo1、Exo2 和 Exo3），这三个基序表征了许多 DNA 聚合酶的 3'-5' 核酸外切结构域。通过定点诱变，我们用丙氨酸或甘氨酸残基取代了每个共有序列中的保守天冬氨酸残基。因此，产生了能够复制线粒体 DNA (mtDNA) 并表现出突变表型的酵母突变体，根据自发性线粒体红霉素抗性突变体的频率增加数百倍来估计。通过将来自 GAL1 启动子的 mtDNA 聚合酶过表达为主要的 140 kDa 多肽，我们表明野生型酶具有错配特异性 3'-5' 核酸外切酶活性。在突变体 D347A 中，这种活性降低了大约 500 倍；相比之下，DNA合成的程度仅略有下降。野生型 mtDNA 聚合酶有效催化单引物 M13 DNA 延伸至全长产物。然而，突变体优先积累低分子量产物。这些数据被扩展到另外两个突变子 D171G 和 D230A。几种聚合酶的 Exo3 位点中存在的 Cys344 残基被甘氨酸取代，产生的突变体具有稍高的 mtDNA 突变率和稍低的 3'-5' 核酸外切活性。我们得出结论，校对是酵母线粒体 DNA 复制准确性的重要决定因素。
The MIP1 gene which encodes yeast mitochondrial DNA polymerase possesses in its N-terminal region the three motifs (Exo1, Exo2 and Exo3) which characterize the 3'-5' exonucleolytic domain of many DNA polymerases. By site directed mutagenesis we have substituted alanine or glycine residues for conserved aspartate residues in each consensus sequence. Yeast mutants were therefore generated that are capable of replicating mitochondrial DNA (mtDNA) and exhibit a mutator phenotype, as estimated by the several hundred-fold increase in the frequency of spontaneous mitochondrial erythromycin resistant mutants. By overexpressing the mtDNA polymerase from the GAL1 promoter as a major 140 kDa polypeptide, we showed that the wild-type enzyme possesses a mismatch-specific 3'-5' exonuclease activity. This activity was decreased by approximately 500-fold in the mutant D347A; in contrast, the extent of DNA synthesis was only slightly decreased. The wild-type mtDNA polymerase efficiently catalyses elongation of singly-primed M13 DNA to the full-length product. However, the mutant preferentially accumulates low molecular weight products. These data were extended to the two other mutators D171G and D230A. Glycine substitution for the Cys344 residue which is present in the Exo3 site of several polymerases generates a mutant with a slightly higher mtDNA mutation rate and a slightly lower 3'-5' exonucleolytic activity. We conclude that proofreading is an important determinant of accuracy in the replication of yeast mtDNA.