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.
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