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

Abstract

The yeast DNA polymerase gamma, Mip1, is a useful tool to investigate the impact of orthologous human disease variants on mitochondrial DNA (mtDNA) replication. However, Mip1 is characterized by a C‐terminal extension (CTE) that is not found on orthologous metazoan DNA polymerases, and the CTE is required for robust enzymatic activity. Two MIP1 alleles exist in standard yeast strains, encoding Mip1[S] or Mip1[Σ]. Mip1[S] is associated with reduced mtDNA stability and increased error rates in vivo. Although the Mip1[S] allele was initially identified in S288c, the Mip1[Σ] allele is widely present among available yeast genome sequences, suggesting that it is the wild‐type (WT) allele. We developed a novel non‐radioactive polymerase gamma assay to assess Mip1 functioning at its intracellular location, the mitochondrial membrane. Membrane fractions were isolated from yeast cells expressing full‐length or CTE truncation variants of Mip1[S] or a chimeric Mip1[S] isoform harboring the Mip1[Σ]‐specific T661 residue (cMip1 T661). Relative incorporation of digoxigenin (DIG)‐11‐deoxyuridine monophosphate (DIG‐dUMP) by cMip1 T661 was higher than that by Mip1[S]. A cMip1 T661variant lacking 175 C‐terminal residues maintained WT levels of DIG‐dUMP incorporation, whereas the C‐terminal variant lacking 205 residues displayed a significant decrease in incorporation. Newly synthesized DIG‐labeled DNA decreased during later phases of reactions carried out at 37°C, suggesting temperature‐sensitive destabilization of the polymerase domain and/or increased shuttling of the nascent DNA into the exonuclease domain. Comparative analysis of Mip1 enzyme functions using our novel assay has further demonstrated the importance of the CTE and T661 encoded by MIP1[Σ] in yeast mtDNA replication.

The Saccharomyces cerevisiae mitochondrial DNA polymerase and its contribution to the knowledge about human POLG‐related disorders

“…As its human counterpart, Mip1 contains an N‐terminal exonuclease domain, a spacer, or linker, domain, a C‐terminal polymerase domain, plus an N‐terminal extension and a C‐terminal extension (Figure 1). Recently, the structural organization of Mip1 was predicted through homology‐based bioinformatic modeling using the crystal structure of human POLG as template, allowing to better define the role of the N‐terminal and C‐terminal extensions 23,24 …”

“…The role of the C‐terminal extension has been analyzed by evaluating the in vitro biochemical properties and the in vivo phenotypes of Mip1 variants with C‐terminal deletions (Table 1). 23,24,33,34 The last 175 amino acids (1,079–1,254) are dispensable for the pol activity and do not affect the maintenance of rho + mtDNA: quite surprisingly, lack of this stretch slightly enhanced the exonuclease activity, resulting in a three‐fold decrease of the mtDNA point mutability, and strongly increased the processivity, likely due to a two‐fold increase in the DNA binding affinity. The following stretch of 21 amino acids (1,058–1,078) contributes to the full polymerase activity but has a minor effect on the exonuclease activity; therefore, the lack of this stretch (together with the last 175 amino acids) strongly increased the petite frequency and slightly the frequency of point mutants resistant to erythromycin (Ery R mutant frequency).…”

“…This substitution is also responsible for about 25% of the petite mutability in strains BY474X 44 . The Ala661 variant showed 50%–70% of the in vitro polymerase activity relative to the Thr661 variant 24 . Structurally, the amino acid 661 is in a linker region typical of the palm subdomain of the subfamily γ of the Family A polymerases.…”

“…Recently, the structural organization of Mip1 was predicted through homologybased bioinformatic modeling using the crystal structure of human POLG as template, allowing to better define the role of the N-terminal and C-terminal extensions. 23,24 Like in most polymerases, the polymerase domain (pol domain, amino acids $388-424 and $585-948) is divided into three subdomains: thumb (388-424 and 585-618 stretches), palm (618-712 and 852-948 stretches) and fingers (712-852 stretch). Three motifs are highly conserved in all eukaryotic Pol γ and are called polA, polB and polC.…”

“…The role of the C-terminal extension has been analyzed by evaluating the in vitro biochemical properties and the in vivo phenotypes of Mip1 variants with C-terminal deletions (Table 1). 23,24,33,34 The last 175 amino acids (1,079-1,254) are dispensable for the pol activity and do not affect the maintenance of rho + mtDNA: quite surprisingly, lack of this stretch slightly enhanced the exonuclease activity, resulting in a three-fold decrease of the mtDNA point mutability, and strongly increased the processivity, likely Abbreviations: ND, not determined; NT, not testable due to respiratory deficient phenotype; RD, respiratory deficient and likely rho 0…”