A compact form of rat liver mitochondrial DNA stabilized by bound proteins

Abstract

A highly folded, rapidly sedimenting form of rat liver mitochondrial DNA has been released from the organelles wiht BRIJ 58 and sodium deoxycholate in the presence of 0.5 M NaCl and isolated by sedimentation velocity in sucrose gradients. Under these conditions a majority of the mitochondrial DNA labeled in vitro sedimented beyond 39 S, the sedimentation coefficient of a highly purified mitochondrial DNA supercoil, and appeared as a stable, heterogeneous population of species ranging in s values between 42 S and about 70 S. Under formamide-spreading conditions most of the rapidly sedimenting forms appeared in the electron microscope as single genome length rosettes constrained at the center in a dense core. Except for an occasional D-loop, no extraordinary structural features were evident along the smooth loops projecting radially from the central core. In sucrose gradients containing various amounts of ethidium bromide, the sedimentation velocity of the folded DNA changed in a biphasic fashion in response to increasing amounts of dye. At a dye concentration of 0.5 microgram per ml the DNA species present reached s value minima, but two major peaks sedimenting at 32 S and 42 S were present at this point. Thus, although these species were similar in superhelix density, there appeared to be additional constraints superimposed upon their tertiary structure that folded these forms to differing degrees of compactness. Direct chemical analyses showed that proteins were bound to the folded DNA at a protein to DNA ratio of about 0.3. Separation of the bound proteins on SDS-polyacrylamide gels revealed an array of proteins ranging in molecular weight between 11,000 and 150,000. Several of the lower molecular weight proteins co-migrated with proteins from the inner mitochondrial membrane, but the major DNA-bound band (Mr = 58,000) was undetectable among the proteins from any other submitchondrial fraction. Digestion of the compact DNA structure with proteinase K under various conditions indicated that the DNA was maintained in the compact conformation by the tightly bound proteins and that the portions of these proteins directly involved in stabilizing the folded DNA were proteinase insensitive unless digestion was carried out in the presence of a disulfide reductant at elevated temperatures.