The rat liver mitochondrial DNA-protein complex: displaced single strands of replicative intermediates are protein coated.
The rat liver mitochondrial DNA-protein complex: displaced single strands of replicative intermediates are protein coated.
复制标题
大鼠肝脏线粒体 DNA-蛋白质复合物：复制中间体的置换单链被蛋白质包被。
DOI：
10.1083/jcb.100.1.251
复制
发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Pavco,PA
中科院分区：
文献类型：
--
作者：
VanTuyle,GC;Pavco,PA
线粒体DNA（mtDNA）-蛋白质复合物经十二烷基硫酸钠裂解后从细胞器中释放出来，用PhenyI-SepharoseCL-4 B柱层析纯化。线粒体DNA结合蛋白P16是复合物中唯一可检测到的蛋白质。用蛋白酶K或枯草杆菌蛋白酶处理复合物，发现存在一个蛋白酶不敏感的亚分子结构域（M，~ 6，000），它保留了与DNA牢固结合的能力。CsCl等密度梯度离心分析化学固定复合物表明，P16绑定到一个大的mtDNA亚群丰富的位移环（D-环）。根据复合物在CsCl梯度中的有效浮力密度以及P16和mtDNA的分子量，估计每个mtDNA平均结合49个P16分子。对于该测量，忽略了在不同CsCl浓度下蛋白质和DNA水合的变化。分析限制性内切酶消化复合物的玻璃纤维过滤器，只结合蛋白质相关的DNA导致保留一个单一的片段，无论酶，或酶，使用。在每种情况下，保留的片段是含有D环的片段。用直接电子显微镜，该蛋白很容易在D-环和扩展D-环的移位单链部分上可视化。核蛋白纤维的直径约为12 nm（不校正钨涂层的厚度），约为相应D环结构双链段长度的1/3。此外，偶见具有缺口环特征的分子显示核蛋白原纤维，推测含有单链缺口片段，连接双链DNA的末端。在所有复合物的双链部分均未发现P16，许多实验室的证据表明，动物mtDNA的复制起点区域与蛋白质成分有关。例如，从HeLa细胞线粒体的Triton X-100裂解物中分离出mtDNA-蛋白质复合物（1）。含有这种结构的限制性内切酶片段的电子显微镜检查揭示了在这些复合物的置换环（D-loop）LA部分附近存在膜样补丁，保留了较小的蛋白质结构
Mitochondrial DNA (mtDNA)-protein complexes were released from the organelles by sodium dodecyl sulfate-lysis and purified by PhenyI-Sepharose CL-4B chromatography. The mitochondrial DNA-binding protein P16 was the only detectable protein in the complex. Treatment of the complex with proteinase K, or subtilisin, revealed the presence of a proteaseinsensitive, submolecular domain (M,~ 6,000) that retained the capacity to bind tenaciously to the DNA. Analysis of chemically fixed complexes by CsCI isopycnic gradient centrifugation showed that P16 was bound to a large subpopulation of mtDNA enriched in displacement loops (D-loops). Based upon the effective buoyant density of the complex in CsCl gradients and the molecular weights of P16 and mtDNA, it was estimated that a mean of 49 P16 molecules were bound per mtDNA. For this measurement, the variation in hydration of protein and DNA at different CsCI concentrations was ignored. Analysis of restriction endonucleasedigested complexes by glass fiber filters that bind only protein-associated DNA resulted in the retention of a single fragment regardless of the enzyme, or enzymes, used. In each case, the retained fragment was the D-loop-containing fragment. With direct electron microscopy, the protein was readily visualized on the displaced single strand portions of D-loops and expanding D-loops. The nucleoprotein fibers were~ 12 nm in diameter without correcting for the thickness of tungsten coating and roughly 1/3 the length of the double strand segment of the corresponding D-loop structure. In addition, occasional molecules with the characteristics of gapped circles were seen exhibiting a nucleoprotein fibril, presumably containing the single strand gap segment, linking the ends of double strand DNA. P16 was not seen on the double strand portions in any of the complexes.Evidence from a number of laboratories suggests that the region of the origin of replication of animal mtDNA is associated with a proteinaceous component. For example, an mtDNA-protein complex was isolated from Triton X-100-lysates of HeLa cell mitochondria (1). Electron microscopic examination of restriction endonuclease fragments containing this structure revealed the presence of a membrane-like patch in the vicinity of the displacement loop (D-loop) LA fraction of these complexes retained a smaller proteinaceous structure