Characterization of a rat liver mitochondrial DNA-protein complex. Replicative intermediates are protected against branch migrational loss.
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.
D000818:Animals; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004272:DNA, Mitochondrial / Q000302:isolation & purification / Q000378:metabolism*; D015247:Deoxyribonuclease HindIII; D003852:Deoxyribonucleoproteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D008930:Mitochondria, Liver / Q000378:metabolism*; D009698:Nucleoproteins / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats
D004272:DNA, Mitochondrial; D003852:Deoxyribonucleoproteins; D009698:Nucleoproteins; D004262:DNA Restriction Enzymes; D015247:Deoxyribonuclease HindIII