The carboxyl-terminal extension on fungal mitochondrial DNA polymerases: identification of a critical region of the enzyme from Saccharomyces cerevisiae.
Fungal mitochondrial DNA (mtDNA) polymerases, in comparison to their metazoan counterparts, harbour unique carboxyl-terminal extensions (CTEs) of varying lengths and unknown function. To determine the essential regions of the 279 residue CTE of the yeast enzyme (Mip1p), several CTE-truncation variants were expressed in Saccharomyces cerevisiae. The respiratory competence of mip1delta175 cells, in which Mip1p lacks the C-terminal 175 residues, is indistinguishable from that of wild-type. In contrast, strains harbouring Mip1pdelta351 and Mip1pdelta279 rapidly lose mtDNA. Approximately one in six mip1delta216 transformants grew on glycerol, albeit poorly. Fluorescence microscopy and Southern blot analysis revealed lower levels of mtDNA in these cells, and the rapid loss of mtDNA during fermentative, but not respiratory, growth. Therefore, only the polymerase-proximal segment of the Mip1p CTE is necessary for mitochondrial function. Comparison of this essential segment with the sequences of other fungal mtDNA polymerases revealed novel features shared among the mtDNA polymerases of the Saccharomycetales.
Young
Matthew J
MJ
Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada R3T 2N2.
Theriault
Steven S
SS
Li
Mingyi
M
Court
Deborah A
DA
D000595:Amino Acid Sequence; D001483:Base Sequence; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D029701:Saccharomyces cerevisiae Proteins; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; C068352:MIP1 protein, S cerevisiae