PACKAGING AND REPLICATION OF MITOCHONDRIAL DNA

拟议研究的目标是继续研究两种不同的 大鼠肝脏线粒体DNA的包装形式。这两种形式包括 (1)可分离的复制静止的mtdna。 在其高S值的基础上，以及(2)复制到其上的分子 结合单一多肽物种(P16)(Mr=16,000)的比率为at 每个mtDNA分子至少有60个多肽。的基本特征 复制中间体是组成新生链的高度 稳定在亲本断裂期间的枝条迁移损失 思特斯。这两种形式的线粒体DNA将通过温和的裂解和释放 根据沉积速度进行分离。每一个物种都将是 通过疏水、分子筛从任何未结合的污染物中提纯 柱层析，琼脂糖凝胶电泳法监测。 这些复合体的超微结构将用电子手段进行检查。 显微镜。线粒体DNA的紧凑形式将通过化学和 以酶法测定其RNA、脂肪、蛋白质和 多胺。非DNA组分在细胞稳定中的作用 紧凑的结构将建立起来。线粒体DNA-P16复制 将通过化学交联研究进一步研究络合物，以 泄露有关结合的P16的相对并置的信息 分子。有效地使用玻璃纤维将过滤器捆绑在一起 方法来选择性地检索P16结合的圆以及特定的 限制，代表显性结合的mtDNA片段 序列。P16将从线粒体裂解物中大量分离出来， 已知含有过量的P16，并通过分子筛分和纯化 制备性等电聚焦。然后，纯净的蛋白质将被 与脱蛋白DNA重组以深入检测 结合相互作用和建立功能关系 在P16和针对分支的复制环路的稳定性之间 迁移。

The goal of the proposed research is to continue the study of two different packaged forms of rat liver mitochondrial DNA. These two forms consist of (1) a replicatively quiescent compact from of mtDNA that can be separated on the basis of its high s value, and (2) replicating molecules to which is bound a single polypeptide species (P16) (MR = 16,000) in a ratio of at least 60 polypeptides per mtDNA molecule. The essential feature of the replictive intermediates is that the constituent nascent strands are highly stabilized against branch migratonal loss during scission of the parental strands. These two forms of the mtDNA will be released by gentle lysis and separated on the basis of sedimentation velocity. Each species will be purified from any unbound contaminants by hydrophobic, molecular sieving column chromatography and monitored by agarose gel electrophoresis. Ultrastructural aspects of the complexes will be examined by electron microscopy. The compact form of mtDNA will be probed chemically and enzymatically to determine its content of RNA, lipid, protein, and polyamines. The role of the non-DNA costituents in the stabilization of the compact structure will be established. The mtDNA-P16 replication complexes will be further examined by chemical crosslinking studies to divulge information regarding the relative juxtaposition of the bound P16 molecules. Effective use will be made of a glass fiber filter binding method to selectively retrieve P16-bound circles as well as specific restriction, fragments of mtDNA that represent the dominant binding sequences. The P16 will be isolated in bulk from mitochondrial lysates, known to contain excess P16, and purified by molecular sieving and preparative isoelectric focusing. The pure protein will then be reconstituted with deproteinized DNA to examine in depth the selectivity of the binding interaction and to establish the functional relationship between P16 and the stability of the replication loops against branch migration.