DNA synthesis in isolated mitochondrial nucleoids from plasmodia of Physarum polycephalum
DNA synthesis in isolated mitochondrial nucleoids from plasmodia of Physarum polycephalum
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DOI：
10.1007/bf01279480
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发表时间：
1998-01-01
期刊：
影响因子：
2.9
通讯作者：
Kuroiwa, T
中科院分区：
文献类型：
--
作者：
Sasaki, N;Sakai, A;Kuroiwa, T
线粒体在线粒体类核（mt-nucleoid，线粒体核的同义词）中含有多个拷贝的线粒体DNA（mtDNA）。线粒体复制子簇（mitochondrial replicon clusters，简称MRC）是一种由线粒体复制子簇（mitochondrial replicon clusters，简称MRCs）组成的线粒体复制子簇。在这项研究中，我们从多头绒泡菌的原质团中分离出结构完整的线粒体类核，并表征了分离的线粒体类核中的DNA合成。通过用0.5% Nonidet P-40溶解高度纯化的线粒体的膜来分离mt-类核。通过观察mt-类核的棒状和MRC的结构来确定分离的mt-类核的结构完整性。分离的mt-类核苷酸需要四个脱氧核苷三磷酸和MgCl 2的DNA合成。DNA合成对阿非迪霉素有抗性，对N-乙基马来酰亚胺和ddTTP的敏感性较低，表明DNA合成是由植物型线粒体DNA聚合酶催化的。在分离的线粒体-类核DNA合成的能力是相似的，在分离的线粒体，尽管去除了大部分的线粒体基质和膜。此外，在体外的DNA合成位点的可视化显示，在每个MRC中发生在分离的线粒体类核DNA合成。这些结果表明，所分离的线粒体类核能够在体外进行有效和系统的DNA合成。因此，使用分离的线粒体类核苷酸应允许在MRC中的mtDNA复制的分子机制的体外表征。
A mitochondrion contains multiple copies of mitochondrial DNA (mtDNA) in the mitochondrial nucleoid (mt-nucleoid, synonym for mitochondrial nuclei). Replicaton of mtDNA in the mt-nucleoids appears to be regulated within groups of adjacent mtDNA molecules, known as mitochondrial replicon clusters (MRCs). In this study, we isolated structurally intact mt-nucleoids from the plasmodia of Physarum polycephalum and characterized DNA synthesis in the isolated mt-nucleoids. The mt-nucleoids were isolated by dissolving the membranes of highly purified mitochondria with 0.5% Nonidet P-40. The structural integrity of the isolated mt-nucleoid was determined by observing the rod shape of the mt-nucleoid and the structure of the MRC. The isolated mt-nucleoids required four deoxyribonucleoside triphosphates and MgCl2 for DNA synthesis. The DNA synthesis was resistant to aphidicolin and showed only low sensitivity to N-ethylmaleimide and to ddTTP, suggesting that the DNA synthesis is catalyzed by plant-type mitochondrial DNA polymerase. The capacity for DNA synthesis in the isolated mt-nucleoids was similar to that in the isolated mitochondria, despite removal of most of the mitochondrial matrix and membrane. Furthermore, visualization of sites of DNA synthesis in vitro revealed that DNA synthesis in the isolated mt-nucleoids occurred in each MRC. These results suggest that the isolated mt-nucleoids are capable of efficient and systematic DNA synthesis in vitro. Therefore, the use of isolated mt-nucleoids should permit in vitro characterization of the molecular mechanism of mtDNA replication in the MRC.