In vivo double-strand breaks occur at recombinogenic G + C-rich sequences in the yeast mitochondrial genome.

Abstract

An optional 46-base-pair G+C-rich element (GC cluster) in the coding region of the yeast mitochondrial varl gene inserts preferentially in crosses into recipient alleles that lack the sequence. Unlike a similar gene conversion event involving the insertion of an optional 1143-base-pair intron, the mitochondrial 21S rRNA gene, which requires the action of a protein encoded by a gene within that intron, conversion of the var) GC cluster does not require any protein product of the mitochondrial genome. We have detected double-strand breaks in the varl gene in mitochondrial DNA isolated from unmated haploid p+ and p-strains at or near the boundaries of the optional GC cluster, as well as at a conserved copy of that sequence 160 base pairs upstream. No double-strand breaks were detected in the recipient varl DNA molecules in the vicinity of the optional GC cluster target sequence. This contrasts with 21S rRNA-encoding DNA (rDNA) intron conversion where the recipient, but not the donor DNA, is cleaved at the element insertion site. These results suggest that although the 21S rDNA intron and the var) GC duster are preferentially inserted into their respective short alleles, these conversions probably occur by different mechanisms.The -80-kilobase (kb) mitochondrial genome of Saccharomyces cerevisiae contains two distinct classes of optional DNA sequences. One class is evident among yeast strains as a variable number of introns located within the genes encoding cytochrome b, cytochrome oxidase subunit I, and the large (21S) rRNA (1); the other class is made up of a large number (>150) of G+C-rich sequences, called GC clusters, most of which are 30-60 base pairs (bp) long and are found dispersed throughout the yeast mitochondrial genome, mostly in the intergenic spaces (2-4). These GC clusters have been subdivided into various groups based on primary sequence homologies (4), and except for those few GC clusters located within the coding sequences of genes, their functions are unknown.For two particular optional mtDNA sequences, specialized recombination is evident in which an allele lacking that sequence acquires it in crosses by unidirectional gene conversion (5). One such recombination involves an optional 1143-bp intron, called co, located within the 21S rRNA gene.In crosses between wt and w-strains, nearly all of the calleles are converted to w+ (6, 7). The mechanism of this recombination event is now partly understood and closely resembles mating type interconversion in yeast nuclear DNA (8). Intron insertion is mediated in part by a protein, called fiti, encoded entirely within the 1143-bp intron (9, 10).That protein is a sequence-specific endonuclease that catalyzes a double-strand break in the DNA of the w-allele at the site of intron insertion (11); conversion probably occurs as a result of the replicative repair of the double-strand break in which the DNA of the a+ allele is used as a template (6-9). This conversion event thus has a clear dependence on mitochondrial protein synthesis.The other specializ...

In vivo rearrangement of mitochondrial DNA in Saccharomyces cerevisiae.

“…Likewise deletion of the symposed repeat has proceeded by means of two other copies of a 49-bp G + C cluster. The involvement of G+C-rich repeats as preferential sites of recombination in mtDNA is in accord with observations from other studies (16,17). Preference for G+C repeats is noteworthy because A+T repeats are more prevalent in basebiased sectors of mtDNA (18).…”

Transcription-Dependent DNA Transactions in the Mitochondrial Genome of a Yeast Hypersuppressive Petite Mutant

“…That the DSBs detected in this study occur around GC cluster C may not be surprising, since GC clusters of several sequence types in yeast mtDNA have been found to be recombinogenic (6,13,49). Further work is required to determine whether the transcription-dependent DSBs we have observed are correlated with recombination structures.…”

“…The role of DSBs in recombination initiation is well documented for both nuclear and mtDNA in yeast cells (17,35,41,49). It is tempting to speculate that the breaks detected in ori1 represent early intermediates of recombination taking place in the HS mtDNA genome.…”

Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria

“…In addition, ori/rep elements do not represent an evolutionary conserved feature required for mtDNA replication (19,20) and they may originate from a mobile or selfish element that invaded and was amplified in the mitochondria of the genus Saccharomyces (21). As the G ϩ C clusters in S. cerevisiae mtDNA represent recombinational hot spots (22,23), the ori/rep elements may serve to promote recombination-dependent replication (RDR) and active transcription could play a stimulatory role in this process (24). Several lines of evidence indicate that rho Ϫ genomes use different mechanisms for their maintenance as the replication of the rho ϩ genomes requires factors like the recombination protein Mgm101, the helicase Hmi1, and the RNA polymerase Rpo41, all of which are dispensable for the replication of certain classes of rho Ϫ genomes (17,25,26).…”