The carboxyl-terminal extension on DNA polymerase a of Saccharomyces cerevisiae is required for mitochondrial DNA maintenance at 37C

Abstract

The mitochondrial DNA (mtDNA) polymerase of 'Saccharomyces ', Mip1p, contains a unique carboxyl-terminal extension (CTE) that extends about 300 amino acids past the last conserved motif in the polymerase domain. To analyze the function of the CTE, we created mutant cell lines expressing versions of the mtDNA polymerase lacking 351, 297, 216, or 176 C-terminal amino acids of the CTE. These mutant cells grew normally at 30C on fermentable and non-fermentable carbon-sources. However, at 37C, during growth on glucose, cells expressing the truncated version of the polymerase, Mip1p[Delta]351, rapidly lost respiratory ability, while Mip1p[Delta]297 cells gradually became respiratory-incompetent. This loss occurred concomitantly with the loss of mtDNA, and was most pronounced in 'MIP1'[Delta]351 cells. ' In organello' replication assays indicate that loss of mtDNA was due to lack of replication competence at 37C. Together, these results indicate that the amino-terminal one-half of the CTE is required for the maintenance of a functional mtDNA polymerase at 37C.