Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes ofHealth, Research, Triangle Park, NC 27709, USA.
Hum Mol Genet. 2010 Jun 1;19(11):2123-33. doi: 10.1093/hmg/ddq089. Epub 2010 Feb 25.
DNA polymerase gamma (pol gamma) is responsible for replication and repair of mitochondrial DNA (mtDNA). Over 150 mutations in POLG (which encodes pol gamma) have been discovered in patients with mitochondrial disorders including Alpers, progressive external ophthalmoplegia and ataxia-neuropathy syndrome. However, the severity and dominance of many POLG disease-associated mutations are unclear, because they have been reported in sporadic cases. To understand the consequences of pol gamma disease-associated mutations in vivo, we identified dominant and recessive changes in mtDNA mutagenesis, depletion and mitochondrial dysfunction caused by 31 mutations in the conserved regions of the gene, MIP1, which encodes the Saccharomyces cerevisiae ortholog of human pol gamma. Twenty mip1 mutant enzymes were shown to disrupt mtDNA replication and may be sufficient to cause disease. Previously uncharacterized sporadic mutations, Q308H, R807C, G1076V, R1096H and S1104C, caused decreased polymerase activity leading to mtDNA depletion and mitochondrial dysfunction. We present evidence showing a limited role of point mutagenesis by these POLG mutations in mitochondrial dysfunction and disease progression. Instead, most mitochondrial defective mip1 mutants displayed reduced or depleted mtDNA. We also determined that the severity of the phenotype of the mip1 mutant strain correlates with the age of onset of disease associated with the human ortholog. Finally, we demonstrated that increasing nucleotide pools by overexpression of ribonucleotide reductase (RNR1) suppressed mtDNA replication defects caused by several dominant mip1 mutations, and the orthologous human mutations revealed severe nucleotide binding defects.
DNA 聚合酶 γ（pol γ）负责线粒体 DNA（mtDNA）的复制和修复。在患有线粒体疾病的患者中，已经发现超过 150 种 POLG（编码 pol γ）突变，包括 Alpers、进行性眼外肌麻痹和共济失调神经病综合征。然而，许多 POLG 疾病相关突变的严重程度和显性程度尚不清楚，因为它们在散发病例中已有报道。为了了解体内 pol γ 疾病相关突变的后果，我们鉴定了 31 种突变在 mtDNA 诱变、耗竭和线粒体功能障碍方面的显性和隐性变化，这些突变位于基因的保守区域 MIP1 中，MIP1 编码人类 pol γ 的酿酒酵母同源物。结果表明，20 种 mip1 突变酶破坏 mtDNA 复制，可能足以引起疾病。以前未表征的散发性突变 Q308H、R807C、G1076V、R1096H 和 S1104C 导致聚合酶活性降低，导致 mtDNA 耗竭和线粒体功能障碍。我们提供的证据表明，这些 POLG 突变导致的点突变在线粒体功能障碍和疾病进展中的作用有限。相反，大多数线粒体缺陷 mip1 突变体显示出减少或耗尽的 mtDNA。我们还确定 mip1 突变菌株表型的严重程度与人类同源物相关疾病的发病年龄相关。最后，我们证明通过过量表达核糖核苷酸还原酶（RNR1）增加核苷酸池可以抑制几种显性 mip1 突变引起的 mtDNA 复制缺陷，并且同源人类突变显示出严重的核苷酸结合缺陷。
