Mhr1p-dependent Concatemeric Mitochondrial DNA Formation for Generating Yeast Mitochondrial Homoplasmic Cells

Abstract

Mitochondria carry many copies of mitochondrial DNA (mtDNA), but mt-alleles quickly segregate during mitotic growth through unknown mechanisms. Consequently, all mtDNA copies are often genetically homogeneous within each individual ("homoplasmic"). Our previous study suggested that tandem multimers ("concatemers") formed mainly by the Mhr1p (a yeast nuclear gene-encoded mtDNA-recombination protein)-dependent pathway are required for mtDNA partitioning into buds with concomitant monomerization. The transmission of a few randomly selected clones (as concatemers) of mtDNA into buds is a possible mechanism to establish homoplasmy. The current study provides evidence for this hypothesis as follows: the overexpression of MHR1 accelerates mt-allele-segregation in growing heteroplasmic zygotes, and mhr1-1 (recombination-deficient) causes its delay. The mt-allele-segregation rate correlates with the abundance of concatemers, which depends on Mhr1p. In G1-arrested cells, concatemeric mtDNA was labeled by [ 14 C]thymidine at a much higher density than monomers, indicating concatemers as the immediate products of mtDNA replication, most likely in a rolling circle mode. After releasing the G1 arrest in the absence of [ 14 C]thymidine, the monomers as the major species in growing buds of dividing cells bear a similar density of 14 C as the concatemers in the mother cells, indicating that the concatemers in mother cells are the precursors of the monomers in buds.

DNA Recombination-Initiation Plays a Role in the Extremely Biased Inheritance of Yeast [rho−] Mitochondrial DNA That Contains the Replication Origin ori5

“…We previously showed that mtDNA concatemers are obligatory intermediates of the partitioning of [rho ϩ ] and [rho Ϫ ] mtDNA and that concatemer formation is carried out primarily by Mhr1-dependent rolling-circle replication (29,30). In this study, we have shown that Ntg1, in addition to Mhr1, is required for the full expression of hypersuppressiveness by HS [ori5] [rho Ϫ ] mtDNA (Fig.…”

“…In our previous studies, we showed by both one-and two-dimensional electrophoresis that the levels of the dense, smeared bands and the arcs correlated positively with the intracellular activity of the Mhr1 protein, which has no DNase activity, indicating that the signals reflected the homologous pairing activity of Mhr1. A straightforward explanation of these previous observations is that the smeared bands and arcs represent concatemers that are formed by Mhr1-dependent rollingcircle replication and this explanation was supported by label-and-chase experiments (29). Our previous studies revealed the reproducibility of the relative amounts of mtDNA concatemers and monomers, and these results were independent of one-or two-dimensional gel electrophoresis (29).…”

“…A straightforward explanation of these previous observations is that the smeared bands and arcs represent concatemers that are formed by Mhr1-dependent rollingcircle replication and this explanation was supported by label-and-chase experiments (29). Our previous studies revealed the reproducibility of the relative amounts of mtDNA concatemers and monomers, and these results were independent of one-or two-dimensional gel electrophoresis (29). Therefore, these results are not a consequence of the uncontrolled degradation of mtDNA species during preparation, and mtDNA species are not lost during the second phase of two-dimensional electrophoresis.…”

“…Label-and-chase experiments and genetic analysis support the conclusion that the concatemers are formed primarily by an Mhr1-dependent rolling-circle mode of replication and that, during or immediately after their transmission into buds, the concatemers are processed into circular mtDNA monomers (29). In addition, Mhr1 plays an active role in vegetative segregation to generate homoplasmic cells, which can be explained by the selective inheritance of an mtDNA clone as concatemers formed by rolling-circle replication on a few selected templates (29). Since Mhr1-dependent rolling-circle replication is the major pathway of mtDNA inheritance, we supposed that Mhr1 functions in hypersuppressiveness.…”

“…Two-dimensional gel electrophoresis analysis was carried out as previously described (8,29). The first dimension was run for 6 h at 1 V/cm on a 0.4% agarose gel in TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.0) at 23°C.…”