Gérard Faye

Physical and genetic organization of petite and grande yeast mitochondrial DNA: IV.† In vivo Transcription Products of Mitochondrial DNA and Localization of 23 S Ribosomal RNA in Petite Mutants of Saccharomyces cerevisiae☆

Abstract A series of isonuclear cytoplasmic petite (ϱ−) mutants presenting various patterns of large deletions and retentions in the C321R — E514R region was used to study the functional organization of mitochondrial DNA. In vivo transcription products of ϱ− mitochondria were analysed by gel electrophoresis and, in an attempt to locate the 23 S ribosomal RNA gene in the mitochondrial genome, by RNA-DNA hybridization. The clones carrying the CR and EP genetic markers contained the 23S mitochondrial rRNA gene and transcribed it in vivo. The clones keeping only the ER marker appeared to have at least a partial sequence of the 23 S rRNA gene; in one clone the whole 23 S rRNA sequence was still present and correctly transcribed. The clones keeping only the CR marker region also had a part of the region sequence complementary to 23 S RNA. No ϱ− clone, in this series, possessed a DNA sequence corresponding to the 16 S rRNA. The possibility that the drug-resistance mutation E514R might be in the 23 S rRNA gene is discussed.

The 3' to 5' exonuclease activity located in the DNA polymerase delta subunit of Saccharomyces cerevisiae is required for accurate replication.

In Saccharomyces cerevisiae, DNA polymerase delta (POLIII), the product of the CDC2 (POL3) gene, possesses, in its N‐terminal half, the well conserved 3‐domain 3′ to 5′ exonuclease site. Strains selectively mutagenized in this site display a mutator phenotype detected as a drastically increased spontaneous forward mutation rate to canavanine resistance or as an elevated reversion rate to lysine prototrophy. Assays on a partially purified extract of the mutant giving the largest mutator effect indicate that the 3′ to 5′ exonuclease activity is reduced below the detection limit whereas the DNA polymerizing activity has wild‐type level. Therefore, our results provide experimental support for the hypothesis that the exonucleolytic proofreading activity associated with DNA polymerase delta resides on the DNA polymerase delta subunit and enhances the fidelity of DNA replication in yeast.

Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III

Saccharomyces cerevisiae cdc2 mutants arrest in the S‐phase of the cell cycle when grown at the non‐permissive temperature, implicating this gene product as essential for DNA synthesis. The CDC2 gene has been cloned from a yeast genomic library in vector YEp13 by complementation of a cdc2 mutation. An open reading frame coding for a 1093 amino acid long protein with a calculated mol. wt of 124,518 was determined from the sequence. This putative protein shows significant homology with a class of eukaryotic DNA polymerases exemplified by human DNA polymerase alpha and herpes simplex virus DNA polymerase. Fractionation of extracts from cdc2 strains showed that these mutants lacked both the polymerase and proofreading 3′‐5′ exonuclease activity of DNA polymerase III, the yeast analog of mammalian DNA polymerase delta. These studies indicate that DNA polymerase III is an essential component of the DNA replication machinery.

Ssu72 is a phosphatase essential for transcription termination of snoRNAs and specific mRNAs in yeast

Ssu72 is an essential yeast protein that is involved in transcription. It physically interacts with transcription initiation and termination complexes. In this report, we provide evidence that Ssu72 is a phosphatase that physically interacts with the CTD kinase Kin28 and functionally interacts with the CTD phosphatase Fcp1. A genome‐wide expression analysis of mutant ssu72‐ts69 during growth in complete medium revealed a number of defects, including the accumulation of a limited number of mRNAs and the read‐through transcription of small nucleolar RNAs and of some mRNAs. We hypothesize that Ssu72 plays a key role in the transcription termination of certain transcripts, possibly by promoting RNA polymerase pausing and release. The possibility that the CTD of the largest subunit of RNA polymerase II is a substrate of Ssu72 is discussed.

The MSS51 gene product is required for the translation of the COX1 mRNA in yeast mitochondria

SummaryThe MSS51 gene product has been previously shown to be involved in the splicing of the mitochondrial pre-mRNA of cytochrome oxidase subunit I (COX1). We show here that it is specifically required for the translation of the COX1 mRNA. Furthermore, the paromomycin-resistance mutation (Pinf454supR) which affects the 15 S mitoribosomal RNA, interferes, directly or indirectly, with the action of the MSS51 gene product. Possible roles of the MSS51 protein on the excision of COX1 introns are discussed.

KIN28, a yeast split gene coding for a putative protein kinase homologous to CDC28.

We have isolated, in yeast, a nuclear gene named KIN28 which presents significant sequence homology with the cell‐division‐cycle CDC28 gene, with members of the protein‐tyrosine kinase family (src, erb, abl, epidermal growth factor, etc.) and those of the family of protein kinases phosphorylating serine and threonine. This strongly suggests that KIN28 is endowed with a protein kinase activity. In contrast with CDC28, KIN28 is interrupted by an intervening sequence. The KIN28 gene failed to complement cdc28 mutations and was shown to be essential for cell proliferation.

Oxygen metabolism and reactive oxygen species cause chromosomal rearrangements and cell death

The absence of Tsa1, a key peroxiredoxin that functions to scavenge H2O2 in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations including gross chromosomal rearrangements (GCRs). Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD6 and several key genes involved in DNA double-strand break repair. In the present study we investigated the causes of GCRs and cell death in these mutants. tsa1-associated GCRs were independent of the activity of the translesion DNA polymerases ζ, η, and Rev1. Anaerobic growth reduced substantially GCR rates of WT and tsa1 mutants and restored the viability of tsa1 rad6, tsa1 rad51, and tsa1 mre11 double mutants. Anaerobic growth also reduced the GCR rate of rad27, pif1, and rad52 mutants, indicating a role of reactive oxygen species in GCR formation in these mutants. In addition, deletion of TSA1 or H2O2 treatment of WT cells resulted in increased formation of Rad52 foci, sites of repair of multiple DNA lesions. H2O2 treatment also induced the GCRs. Our results provide in vivo evidence that oxygen metabolism and reactive oxygen species are important sources of DNA damages that can lead to GCRs and lethal effects in S. cerevisiae.

A Newly Identified Essential Complex, Dre2-Tah18, Controls Mitochondria Integrity and Cell Death after Oxidative Stress in Yeast

A mutated allele of the essential gene TAH18 was previously identified in our laboratory in a genetic screen for new proteins interacting with the DNA polymerase delta in yeast [1]. The present work shows that Tah18 plays a role in response to oxidative stress. After exposure to lethal doses of H2O2, GFP-Tah18 relocalizes to the mitochondria and controls mitochondria integrity and cell death. Dre2, an essential Fe/S cluster protein and homologue of human anti-apoptotic Ciapin1, was identified as a molecular partner of Tah18 in the absence of stress. Moreover, Ciapin1 is able to replace yeast Dre2 in vivo and physically interacts with Tah18. Our results are in favour of an oxidative stress-induced cell death in yeast that involves mitochondria and is controlled by the newly identified Dre2-Tah18 complex.

Physical and genetic organization of petite and grande yeast mitochondrial DNA: III. High resolution melting and reassociation studies☆

Mitochondrial DNAs from 13 petite mutants have been analyzed by means of high-resolution melting and reassociation in solution, in an attempt to relate their physical chemical properties to the mitochondrial genotype, which displays various combinations of genetic marker deletions. The kinetic complexities of the petite mtDNAs were found to range from 13 down to 1500 of that of the grande mtDNA; the loss in sequential complexity undergone by petite mtDNAs parallels the loss in mitochondrial genetic complexity. Melting profiles of petite mtDNAs can be resolved into well-defined peaks. Some of them are specific to the marker genes. The genotypic specificity increases as the sequential complexity of mtDNA decreases. The mtDNA region conferring resistance to erythromycin could in this way be shown to be characterized by two melting peaks, at 72 °C and 75 °C. The results are interpreted in terms of selective enrichment in gene-specific sequences.

MSS18, a yeast nuclear gene involved in the splicing of intron aI5 beta of the mitochondrial cox1 transcript.

We have isolated and characterized a nuclear gene, MSS18, which is implicated in the splicing of intron aI5 beta of the mitochondrial cox1 transcript (subunit 1 of the cytochrome c oxidase). Northern blotting and S1 nuclease protection experiments as well as the analysis of mitochondrial point revertants suggest that mss18 mutations block (perhaps indirectly) the cleavage of the 5′ exon‐intron junction of aI5 beta. Mitochondrial point revertants also indicate that up to 13 bases of the aI5 beta exon sequence, upstream of the 5′ splice site of aI5 beta, are involved in vivo in the splicing of this intron. The implications of this result on the splicing of group I introns are discussed.