Characterization of a rat liver mitochondrial DNA-protein complex. Replicative intermediates are protected against branch migrational loss.
Characterization of a rat liver mitochondrial DNA-protein complex. Replicative intermediates are protected against branch migrational loss.
复制标题
大鼠肝脏线粒体 DNA-蛋白质复合物的表征。
DOI：
10.1016/s0021-9258(18)42962-6
复制
发表时间：
1981
期刊：
影响因子：
--
通讯作者：
P. Pavco
中科院分区：
文献类型：
--
作者：
G. V. Van Tuyle;P. Pavco
用十二烷基硫酸钠裂解法从大鼠肝线粒体中释放出一种稳定的DNA-蛋白质复合物，并在蔗糖密度梯度中用沉降速度分离。用0.5M NaCl洗涤mtDNA-蛋白质复合物，并通过羟基磷灰石柱色谱法除去任何未结合的污染物。复合物中唯一可检测的多肽是具有7.6-7.8的弱碱性等电点的单一低分子量物质（Mr = 16，000）。用限制性内切酶HindIII完全消化mtDNA-蛋白质复合物，在琼脂糖凝胶中显示由最大片段群的子集组成的“额外”条带。在这个子集中的片段被证明含有复制的中间体，这是由于在复制环的区域中的亲本链分离而在电泳迁移中被延迟。没有损失的新生链由于分支迁移的亲本链后，HindIII切割的共价闭合的环状DNA的mtDNA-蛋白质复合物。然而，HindIII消化完全脱蛋白的mtDNA导致从复制分子的新生链的定量损失。这些结果被解释为证据表明，在复合物中存在的单一低分子量多肽在保持复制环的完整性在亲本链断裂过程中发挥了重要作用。
A stable DNA-protein complex was released from rat liver mitochondria by sodium dodecyl sulfate-lysis and isolated by sedimentation velocity in sucrose density gradients. The mtDNA-protein complex was washed with 0.5 M NaCl and any unbound contaminants were removed by hydroxyapatite column chromatography. The only detectable polypeptide in the complex was a single low molecular weight species (Mr = 16,000) having a slightly basic isoelectric point of 7.6-7.8. Complete digestion of the mtDNA-protein complex with restriction endonuclease HindIII revealed in agarose gels an “extra” band consisting of a subset of the largest fragment population. The fragments in this subset were shown to contain the replicative intermediates which were retarded in electrophoretic migration due to the parental strand separation in the region of the replication loops. No loss of nascent strands due to branch migration of the parental strands was observed upon HindIII cleavage of the covalently closed circular DNA in the mtDNA-protein complex. However, HindIII digestion of completely deproteinized mtDNA resulted in quantitative loss of nascent strands from replicating molecules. These results are interpreted as evidence that the single low molecular weight polypeptide present in the complex plays a major role in maintaining the integrity of replication loops during parental strand scission.