Abstract
The catalytic subunit (alpha) of mitochondrial DNA polymerase (pol gamma) shares conserved DNA polymerase and 3'-5' exonuclease active site motifs with Escherichia coli DNA polymerase I and bacteriophage T7 DNA polymerase. A major difference between the prokaryotic and mitochondrial proteins is the size and sequence of the region between the exonuclease and DNA polymerase domains, referred to as the spacer in pol gamma-alpha. Four gamma-specific conserved sequence elements are located within the spacer region of the catalytic subunit in eukaryotic species from yeast to humans. To elucidate the functional roles of the spacer region, we pursued deletion and site-directed mutagenesis of Drosophila pol gamma. Mutant proteins were expressed from baculovirus constructs in insect cells, purified to near homogeneity, and analyzed biochemically. We find that mutations in three of the four conserved sequence elements within the spacer alter enzyme activity, processivity, and/or DNA binding affinity. In addition, several mutations affect differentially DNA polymerase and exonuclease activity and/or functional interactions with mitochondrial single-stranded DNA-binding protein. Based on these results and crystallographic evidence showing that the template-primer binds in a cleft between the exonuclease and DNA polymerase domains in family A DNA polymerases, we propose that conserved sequences within the spacer of pol gamma may position the substrate with respect to the enzyme catalytic domains.
Highlights
The mitochondrion is the eukaryotic organelle that carries out oxidative phosphorylation, fulfilling cellular requirements for energy production
Amino Acid Residues Important for DNA Polymerase Activity Are Distributed within the Spacer Region of Pol ␥-␣—To examine the role of the four conserved sequence elements, ␥1–␥4, in the spacer region of the catalytic subunit of Drosophila DNA polymerase ␥, we began by producing recombinant proteins carrying amino acid deletions in each of the conserved elements (Fig. 1)
Mutant pol ␥-␣ variants were co-expressed with wildtype pol ␥- in the baculovirus system, and the reconstituted holoenzymes were purified by phosphocellulose chromatography
Summary
The mitochondrion is the eukaryotic organelle that carries out oxidative phosphorylation, fulfilling cellular requirements for energy production. Disruption of mitochondrial energy metabolism can occur by genetic or biochemical mechanisms and is associated with human disorders including degenerative diseases, cancer, and aging [1]. Mutations in both the mitochondrial genome and in nuclear genes whose products have mitochondrial functions are linked to mitochondrial disease syndromes. The specific function of the conserved spacer sequences is not known, a missense mutation in the spacer region of Drosophila pol ␥-␣ causes mitochondrial and nervous system dysfunction and developmental lethality in the larval third instar [24]. Our results show that mutations in conserved amino acid sequences within the spacer region alter enzyme activity, processivity, and DNA binding affinity. Several mutations affect differentially DNA polymerase and exonuclease activity and/or functional interactions with mitochondrial single-stranded DNA-binding protein, mtSSB
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