Evolution of the Metazoan Mitochondrial Replicase

Abstract

The large number of complete mitochondrial DNA (mtDNA) sequences available for metazoan species makes it a good system for studying genome diversity, although little is known about the mechanisms that promote and/or are correlated with the evolution of this organellar genome. By investigating the molecular evolutionary history of the catalytic and accessory subunits of the mtDNA polymerase, pol γ, we sought to develop mechanistic insight into its function that might impact genome structure by exploring the relationships between DNA replication and animal mitochondrial genome diversity. We identified three evolutionary patterns among metazoan pol γs. First, a trend toward stabilization of both sequence and structure occurred in vertebrates, with both subunits evolving distinctly from those of other animal groups, and acquiring at least four novel structural elements, the most important of which is the HLH-3β (helix-loop-helix, 3 β-sheets) domain that allows the accessory subunit to homodimerize. Second, both subunits of arthropods and tunicates have become shorter and evolved approximately twice as rapidly as their vertebrate homologs. And third, nematodes have lost the gene for the accessory subunit, which was accompanied by the loss of its interacting domain in the catalytic subunit of pol γ, and they show the highest rate of molecular evolution among all animal taxa. These findings correlate well with the mtDNA genomic features of each group described above, and with their modes of DNA replication, although a substantive amount of biochemical work is needed to draw conclusive links regarding the latter. Describing the parallels between evolution of pol γ and metazoan mtDNA architecture may also help in understanding the processes that lead to mitochondrial dysfunction and to human disease-related phenotypes.

Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA

“…Furthermore, it has been demonstrated that the dimeric accessory subunit of HsPol ␥ (Pol ␥␤) enhances the ssDNA binding affinity of Pol ␥␣ by ϳ100-fold and increases its processivity by 50 -100-fold (31,39). Given that the insect Pol ␥ contains a monomeric Pol ␥␤, the differences we observe in their interactions with the template DNA may also reflect differences in the contributions of Pol ␥␤ to the properties of the holoenzyme (17). In that regard, it has been demonstrated that mutations within the dimerization interface of human Pol ␥␤, which is absent in the insect homologue, result in decreased formation of a productive Pol ␥-DNA complex, resulting in a lower processivity of the enzyme (17,40).…”

“…Given that the insect Pol ␥ contains a monomeric Pol ␥␤, the differences we observe in their interactions with the template DNA may also reflect differences in the contributions of Pol ␥␤ to the properties of the holoenzyme (17). In that regard, it has been demonstrated that mutations within the dimerization interface of human Pol ␥␤, which is absent in the insect homologue, result in decreased formation of a productive Pol ␥-DNA complex, resulting in a lower processivity of the enzyme (17,40). Different properties of the D. melanogaster and H. sapiens polymerases ␥ might also be extrapolated to the mtDNA replication process to suggest that although the overall efficiency of DNA synthesis in both systems is probably similar, the mechanism of initiation of DNA strand synthesis may differ.…”

“…The lower activity of DmPol ␥ in the absence of SSB may result from its lower affinity for the template DNA and/or weaker ability to disrupt secondary structural elements within ssDNA template. Indeed, we reported recently a comparative analysis of Pol ␥ sequences from various metazoan taxa that revealed several structural differences between the enzymes of vertebrata and other metazoan taxa, some of which may explain differences in interactions with the template DNA (17 ) in the catalytic subunit of human Pol ␥ (Pol ␥␣), absent in protostome homologs, locate in close proximity to the minor groove of the primer-template DNA modeled at the Pol ␥␣ binding site (38). The precise role of these residues remains to be investigated, but given their location and positive charge, we predict that they enhance the DNA binding affinity of the enzyme.…”

“…We sought to assess the mechanism of the stimulatory effect of human mtSSB on its cognate Pol ␥. We recently published a comparative analysis of Pol ␥ sequences and structural organization from various metazoan taxa (17) and included in this study homologous proteins from D. melanogaster and E. coli to examine similarities and differences between these systems. Our current findings provide evidence of a relationship between Pol ␥ activity and SSB-generated template DNA organization.…”

Animal Mitochondrial DNA Replication

“…The heterotrimeric organization of the human replicase, comprising a single Pol γ-α and two Pol γ-β polypeptides, appears to be common to all vertebrate mtDNA polymerases [18]. Pol γ-α consists of three domains, arranged spatially to interact with the Pol γ-β dimer, and to facilitate the transition between its 5′-3′ polymerase and 3′-5′ exonuclease activities (Figure 2).…”