Amino and carboxy-terminal extensions of yeast mitochondrial DNA polymerase assemble both the polymerization and exonuclease active sites

A non‐radioactive DNA synthesis assay demonstrates that elements of the Sigma 1278b Mip1 mitochondrial DNA polymerase domain and C‐terminal extension facilitate robust enzyme activity

“…However, the result with cMip1 T661‐Δ216 is unexpected, because this enzyme can support mtDNA maintenance under selective conditions (Young et al, 2006), and the purified Mip1[Σ]Δ216 enzyme has ~70% of the specific activity of the full‐length enzyme on calf thymus DNA and is active on singly primed single‐stranded DNA (Viikov et al, 2012). Given the undetectable signal from cMip1 T661‐Δ216 fractions, it was expected that the cMip1 T661‐Δ222‐containing fractions would not show detectable activity; similarly, Mip1[S]Δ227 was demonstrated to be inactive in vitro (Trasvina‐Arenas et al, 2019).…”

“…Cells harboring Mip1[Σ]Δ205 retain mtDNA and respiratory function, whereas those expressing Mip1[Σ]222 do not, indicating the importance of the N‐terminal portion of α2 (residues 1033–1048; see Figure 1b). The importance of Mip1 residues 1028–1038 has been demonstrated with Mip1[Σ]Δ216 and Mip1[S]Δ227 variants in vitro whereby the balance between polymerase and exonuclease activities is skewed in the Mip1[S]Δ227 variant, which synthesizes very little DNA in vitro (Trasvina‐Arenas et al, 2019; Viikov et al, 2012). Therefore, to assess the role of the residues in α2 that distinguish Mip1[Σ]Δ205 and Mip1[Σ]Δ222, mutations were made in the cMip1 T661 ( Mip1[Σ] ‐like) gene to replace codons for residues 1033–1037 in this segment with that for glycine (Figure S5).…”

“…Based on the recently published Mip1 structural homology model, here we define the 1254‐amino acid residue Mip1 polymerase domain to encompass residue Y393 (of the γ1 conserved signature sequence) to D990 (Trasvina‐Arenas et al, 2019). pcMIP1 T661 containing the MIP1 [Σ] polymerase domain sequence (Baruffini et al, 2007) (see Figure S1) was constructed by cutting pMIP1[S] with AvrI I and Pst I to remove a 1539‐bp fragment, followed by recombination in S150 mip1Δ with the corresponding PCR product generated from S150 ( MIP1 [Σ]) genomic DNA.…”

“…Carboxyl‐terminal extensions (CTEs) on fungal mtDNA polymerases were first noted in Saccharomyces (Hu, Vanderstraeten, & Foury, 1995) and Neurospora (Ko T, Bertrand H, Michigan State University, personal communication) and are of variable lengths on enzymes from ascomycetous fungi, including yeast‐like fungi, pyrenomycetes, and plectomycetes (Young et al, 2006). The terminal 216 residues of the CTE of both the Mip1[Σ] and Mip1[S] isoforms have been demonstrated to be essential for respiratory growth in vivo (Trasvina‐Arenas et al, 2019; Young et al, 2006) and are required for robust activity of the recombinant Mip1[S] enzyme purified from Escherichia coli (Viikov, Jasnovidova, Tamm, & Sedman, 2012). In vivo expression of the Mip1 variant lacking the 216 C‐terminal residues (Mip1[Σ]Δ216) causes an increase in mtDNA mutations, a decrease in mtDNA copy number during respiratory growth, and a complete failure to maintain mtDNA during non‐selective, fermentative growth, summarized in Figure 1a (and see Young et al, 2006).…”

“…Pol, mtDNA polymerase activity; Exo, 3′‐5′ DNA exonuclease activity as measured on dsDNA primer templates in the absence of dNTPs; Exo/Pol, exonuclease to polymerase activity ratio on dsDNA in the presence of dNTP; bold upward‐facing arrows indicate detectable exonuclease activity without detectable polymerase activity. Citations are indicated by V from Viikov et al (2012), T from Trasvina‐Arenas et al (2019), Y from Young et al (2006), and C from the current study; Er R , erythromycin resistance; Aff., affinity for template DNA; Proc., processivity; St.Dis., strand displacement. (b) CLUSTALW ( http://align.genome.jp/) alignment of the C‐termini of Mip1 sequences of S150 ( MIP1[Σ] ) and YPH499 ( MIP1[S] ) laboratory strains (see Young & Court, 2008, for details).…”