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Modular architecture and human disease alleles of the mitochondrial replicative DNA helicase
Author(s) -
Kaguni Laurie S.,
Ziebarth Tawn D.,
Matsushima Yuichi,
Farr Carol L.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.111.1
Subject(s) - helicase , biology , mitochondrial dna , rna helicase a , genetics , microbiology and biotechnology , primase , dna , mutant , random hexamer , gene , rna , reverse transcriptase
The human mtDNA helicase shares amino acid sequence and functional similarities with bacteriophage T7 primase‐helicase. mtDNA helicase exists as a hexamer that is destabilized under low salt conditions, but can be maintained by inclusion of Mg 2+ and ATP, and by temperatures above 20 °C. Limited proteolysis reveals distinct N‐ and C‐terminal polypeptides that represent minimal structural domains, and yields several stable fragments that differ in the presence of nucleoside di‐ and triphosphate and DNA. Truncations of the N‐ and C‐termini affect differentially DNA‐dependent ATPase activity, and whereas a C‐terminal domain polypeptide is functional, an N‐terminal domain polypeptide lacks ATPase activity. Molecular analysis of the Drosophila homolog was used to evaluate the physiological consequences of active site and human disease alleles. RNA interference reduces expression of d ‐mtDNA helicase to an undetectable level in Schneider cells, causing an ∼5‐fold decrease in mtDNA copy number, whereas overexpression increases mtDNA levels 1.4 fold. Overexpression of helicase active site mutants results in a severe depletion of mtDNA, and a dominant‐negative lethal phenotype. Interestingly, overexpression of mutants analogous to human disease alleles show differential effects, ranging from dominant negative effects to increases in mtDNA copy number. This research was supported by NIH grant GM45295.