Comparison of replication structures in mutant and wild-type mitochondrial DNA of the yeast Saccharomyces cerevisiae

Abstract

Mitochondrial genomes vary greatly in structure and size from organism to organism. The mechanisms for replicating mitochondrial DNA (mtDNA) are not yet completely understood. Saccharomyces cerevisiae, baker's yeast, is a model organism utilized to study mtDNA replication. An issue arises using it for research since two forms are commonly used: the wild-type form, called rho+, and a form that has undergone massive mtDNA deletions, called rho-. Since rho- cells only contain a fragment of the wild-type mitochondrial genome amplified to an equivalent mass, it is of concern that these strains may replicate in an unusual manner, akin to an SOS response. Using neutral/neutral two-dimensional (2-D) gel electrophoresis, this study presents evidence to support a mtDNA replication mechanism that is common to both rho+ and rho- strains. Both intact and restriction enzyme-digested mitochondrial genomes were examined. The most widely-accept model of mtDNA replication in S. cerevisiae is the strand-asynchronous asymmetric model. This mechanism would generate primarily single-stranded replication intermediates. 2-D gel analysis of rho+ and rho- strains revealed both single-stranded and double-stranded replication intermediates along with potential RNA-dependent species. This suggests that more models of replication are occurring in addition to the strand-asynchronous one. These potential models include a rolling circle mechanism and also one that is dependent on recombination. Recombination-dependent replicating has been previously reported in mammals, but this is the first evidence presented for it in yeast.