Abstract
Mitochondria are the essential energy‐producing organelle in eukaryotes, and represent the only organelle in animals that contains its own genome. Maintenance of the mitochondrial DNA (mtDNA) genome is dependent on myriad nuclear‐encoded proteins including the mtDNA helicase, which is an essential component of the replicative machinery. Human mtDNA helicase shares a high degree of sequence similarity with the bacteriophage T7 primase‐helicase gene 4 protein, and catalyzes duplex unwinding in the 5’‐3’ direction. We have shown that it is an oligomer in solution that is destabilized under low salt conditions. We present here the effects of cofactors on its stability and oligomerization state. We show that the low salt sensitivity of the mtDNA helicase is alleviated by the presence of magnesium, nucleotide and increased temperature. Electron microscopic and glutaraldehyde cross‐linking analyses provide the first evidence of a heptameric oligomer, and its interconversion from a hexameric form. Limited proteolysis by trypsin shows that binding of nucleoside triphosphate produces a conformational change that is distinct from the conformation observed in the presence of nucleotide diphosphate. We find that single‐stranded DNA binding occurs in the absence of cofactors, and renders the mtDNA helicase more susceptible to proteolytic digestion. Experiments to examine oligomeric state upon DNA binding are underway to evaluate the functional form of the enzyme, and its dynamic changes along the reaction pathway. This research was supported by NIH grant GM45295.
Highlights
The human mitochondrial DNA4 helicase represents the most recent addition to the mitochondrial replication fork
Range of 4Ϫ37 °C enhance the recovery of soluble protein Oligomeric States of the Human mitochondrial DNA (mtDNA) Helicase in the Preswhereas at low salt, we observe complete insolubility (Fig. 1, ence of Cofactors and DNA Substrate—To investigate the oligoA–C)
The presence of Mg2ϩ meric state of the human mtDNA helicase in the presence or increases the solubility of the enzyme at both salt concentra- absence of cofactors and DNA under low salt conditions, we tions, and stability is increased further with the addition of used glutaraldehyde cross-linking followed by Sodium dodecyl sulfate (SDS)-PAGE
Summary
The human mitochondrial DNA (mtDNA) helicase represents the most recent addition to the mitochondrial replication fork. The bacteriophage T7 gp protein exists predominantly in the hexameric state, yet both a crystal structure and electron microscopic images demonstrate a heptameric oligomer either in the absence of cofactors, or in the presence of Mg2ϩ-dTDP [3, 4]. T7 gp has been shown to exist as a dimer and trimer that oligomerize into hexamers in the presence of dTTP, but in the absence of Mg2ϩ (14 –16) All of these proteins are members of the same superfamily, different nucleotide requirements for achieving an active oligomeric state most likely serve a regulatory role that is specific to the given organism. Oligomeric States of Human mtDNA Helicase trate numerous conformations of T7 gp in the presence of various nucleotides and DNA. These conformations are presumed to facilitate nucleotide hydrolysis and the mechanism of single-stranded DNA (ssDNA) translocation that leads to double-stranded DNA (dsDNA) unwinding
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