Structure, function and evolution of the animal mitochondrial replicative DNA helicase

Abstract

The mitochondrial replicative DNA helicase is essential for animal mitochondrial DNA (mtDNA) maintenance. Deleterious mutations in the gene that encodes it cause mitochondrial dysfunction manifested in developmental delays, defects and arrest, limited life span, and a number of human pathogenic phenotypes that are recapitulated in animals across taxa. In fact, the replicative mtDNA helicase was discovered with the identification of human disease mutations in its nuclear gene, and based upon its deduced amino acid sequence homology with bacteriophage T7 gene 4 protein (T7 gp4), a bi-functional primase-helicase. Since that time, numerous investigations of its structure, mechanism, and physiological relevance have been reported, and human disease alleles have been modeled in the human, mouse, and Drosophila systems. Here, we review this literature and draw evolutionary comparisons that serve to shed light on its divergent features.

Animal Mitochondrial DNA Replication

“…Evidence suggesting the importance of this putative DNA-binding region includes the findings: 1) expressing D. melanogaster mtDNA helicase bearing analogous mutations found in human patients causes severe mtDNA depletion in S2 cells [34], consistent with the fact that the recombinant N-terminal domain of the insect enzyme binds to both ss and dsDNA [35]; and 2) recombinant human mtDNA helicases lacking part or all of the RPD exhibited lower ssDNA-binding, ATPase and unwinding activities [36], in agreement with the observed decrease in mtDNA copy number in human cultured cells [37]. Interestingly, the RPD of the mtDNA helicase appears to have evolved these novel functions without losing either the polypeptide fold or some of the conserved amino acid residues of a prokaryotic primase, even though it does not synthesize RNA primers [27, 36]. …”

“…Recently, we reviewed comprehensively the literature on the structure, catalytic activity, and evolution of the animal mtDNA helicase, including an evaluation of human pathogenic variants and a discussion of current animal models [27]. Among the important findings, two features of the replicative mtDNA helicase are notable: its remarkable resemblance to the bacteriophage T7 gp4, a bifunctional primase-helicase (see Chapter 3 of this Volume), and the numerous mutations in the human gene associated with mitochondrial disorders.…”

“…Molecular modeling and mapping of the disease-related residues reported to date in the human enzyme identified two major structural regions that we explored in detail. First, the abundance of pathogenic residues found in the Linker region and CTD argues that their effects on subunit interactions are a major cause of mtDNA replication defects leading to human disorders [27]. Oligomerization in replicative helicases is key to the formation of the nucleotide-binding pocket at the protomer-protomer interface that is required for proper positioning of the substrate for hydrolysis, which is coordinated with translocation of the helicase on DNA [29].…”

“…If so, the RPD in mtDNA helicase binds DNA in a configuration not described for T7 gp4 or other prokaryotic primases [27]. Evidence suggesting the importance of this putative DNA-binding region includes the findings: 1) expressing D. melanogaster mtDNA helicase bearing analogous mutations found in human patients causes severe mtDNA depletion in S2 cells [34], consistent with the fact that the recombinant N-terminal domain of the insect enzyme binds to both ss and dsDNA [35]; and 2) recombinant human mtDNA helicases lacking part or all of the RPD exhibited lower ssDNA-binding, ATPase and unwinding activities [36], in agreement with the observed decrease in mtDNA copy number in human cultured cells [37].…”

“…Whereas oligomerization clearly involves residues in the Linker region and CTD, both the oligomeric form and the conformation of individual protomers in the human mtDNA helicase appear to be dynamic [33, 38, 39]. A high proportion of structurally-heterogeneous homohexamers with 3-fold symmetry was observed at high ionic strength with protein purified in the baculovirus expression system, which contrasts with a more homogeneous homoheptameric species with a clear 7-fold symmetry (Figure 3C) observed at low ionic strength and in the presence of Mg 2+ and ATPγS [38].…”