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.