Abstract
The purified endonuclease of bovine heart mitochondria extensively degrades a variety of DNA templates in vitro but shows a remarkably strong preference to nick within one specific evolutionarily conserved sequence block of 12 consecutive guanine residues which resides just upstream from the heavy strand origin of mitochondrial DNA replication (Low, R. L., Cummings, O. W., and King, T. C. (1987) J. Biol. Chem. 262, 16164-16170). If the enzyme serves to provide an important nicking function at this site in vivo, then mitochondrial factors likely exist which further enhance the enzyme's recognition of this locus and prevent cleavage at other less favored sites. In this study, we report that specific membrane phospholipids appear to exert such effects in vitro. In standard endonuclease assays, low levels of phosphatidylcholine or phosphatidylethanolamine (0.5 mM) stimulate the purified enzyme activity 10-20-fold. However, at moderate levels (20-40 mM), these phospholipids largely inhibit widespread degradation of duplex DNA while still allowing site-specific nicking at the conserved guanine target in the mitochondrial genome. These findings suggest that an interaction of the endonuclease with major lipid components of the inner membrane could be an important determinant of the enzyme's specificity for mitochondrial DNA.
Highlights
The purified endonuclease of bovine heart mitochondria extensively degrades a variety of DNA templates in vitro but shows a remarkably strong preference to nick within one specific evolutionarily conserved sequence block of 12 consecutive guanine residues which resides just upstream from the heavy strand origin of mitochondrial
The participation of the endonuclease at this specific site in vivo to serve as the nicking component of a novel swivel is an interesting possibility. This pattern of cleavage of the mitochondrial endonuclease looks similar if not identical to that of a nuclear endonuclease activity called endonuclease G, which in vitro appears to target specific runs of guanine residues found in the regulatory domains of some nuclear genes [16]
Identified in a Crude Fraction of Mitochondrial Protein but Stimulates the Activity of the Purified Enzyme--The large majority (>90%) of the bovine mitochondrial endonuclease resides in a crude concentrate of proteins called Fraction II, which is prepared by ammonium sulfate fractionation of the mitochondrial lysate [1]
Summary
At moderate levels (20-40 mM), these phospholipids largely inhibit widespread degradation of duplex DNA while still allowing site-specific nicking at the conserved guanine target in the mitochondrial genome These findings suggest that an interaction of the endonuclease with major lipid components of the inner membrane could be an important determinant of the enzyme’s specificity for mitochondrial. The participation of the endonuclease at this specific site in vivo to serve as the nicking component of a novel swivel is an interesting possibility This pattern of cleavage of the mitochondrial endonuclease looks similar if not identical to that of a nuclear endonuclease activity called endonuclease G, which in vitro appears to target specific runs of guanine residues found in the regulatory domains of some nuclear genes [16]. Low levels of phosphatidylcholine, for example, stimulate endonucleolytic degradation of duplex DNA, moderate levels prevent extensive DNA fragmentation while still allowing specific nicking of the conserved guanine target of the D-loop region
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