Amino and carboxy-terminal extensions of yeast mitochondrial DNA polymerase assemble both the polymerization and exonuclease active sites.

Human and yeast mitochondrial DNA polymerases (DNAPs), POLG and Mip1, are related by evolution to bacteriophage DNAPs. However, mitochondrial DNAPs contain unique amino and carboxyl-terminal extensions that physically interact. Here we describe that N-terminal deletions in Mip1 polymerases abolish polymerization and decrease exonucleolytic degradation, whereas moderate C-terminal deletions reduce polymerization. Similarly, to the N-terminal deletions, an extended C-terminal deletion of 298 amino acids is deficient in nucleotide addition and exonucleolytic degradation of double and single-stranded DNA. The latter observation suggests that the physical interaction between the amino and carboxyl-terminal regions of Mip1 may be related to the spread of pathogenic POLG mutant along its primary sequence.

Yeast mitochondrial RNA polymerase primes mitochondrial DNA polymerase at origins of replication and promoter sequences.

Three proteins phylogenetically grouped with proteins from the T7 replisome localize to yeast mitochondria: DNA polymerase γ (Mip1), mitochondrial RNA polymerase (Rpo41), and a single-stranded binding protein (Rim1). Human and T7 bacteriophage RNA polymerases synthesize primers for their corresponding DNA polymerases. In contrast, DNA replication in yeast mitochondria is explained by two models: a transcription-dependent model in which Rpo41 primes Mip1 and a model in which double stranded breaks create free 3' OHs that are extended by Mip1. Herein we found that Rpo41 transcribes RNAs that can be extended by Mip1 on single and double-stranded DNA. In contrast to human mitochondrial RNA polymerase, which primes DNA polymerase γ using transcripts from the light-strand and heavy-strand origins of replication, Rpo41 primes Mip1 at replication origins and promoter sequences in vitro. Our results suggest that in ori1, short transcripts serve as primers, whereas in ori5 an RNA transcript longer than 29 nucleotides is used as primer.

Complete mitochondrial genome of Concholepas concholepas inferred by 454 pyrosequencing and mtDNA expression in two mollusc populations.

Despite the great relevance of mitochondrial genome analysis in evolutionary studies, there is scarce information on how the transcripts associated with the mitogenome are expressed and their role in the genetic structuring of populations. This work reports the complete mitochondrial genome of the marine gastropod Concholepas concholepas, obtained by 454 pryosequencing, and an analysis of mitochondrial transcripts of two populations 1000 km apart along the Chilean coast. The mitochondrion of C. concholepas is 15,495 base pairs (bp) in size and contains the 37 subunits characteristic of metazoans, as well as a non-coding region of 330 bp. In silico analysis of mitochondrial gene variability showed significant differences among populations. In terms of levels of relative abundance of transcripts associated with mitochondrion in the two populations (assessed by qPCR), the genes associated with complexes III and IV of the mitochondrial genome had the highest levels of expression in the northern population while transcripts associated with the ATP synthase complex had the highest levels of expression in the southern population. Moreover, fifteen polymorphic SNPs were identified in silico between the mitogenomes of the two populations. Four of these markers implied different amino acid substitutions (non-synonymous SNPs). This work contributes novel information regarding the mitochondrial genome structure and mRNA expression levels of C. concholepas.

High fat diet-induced liver steatosis promotes an increase in liver mitochondrial biogenesis in response to hypoxia.

Mitochondrial DNA (mtDNA) copy number plays a key role in the pathophysiology of metabolic syndrome-related phenotypes, but its role in non-alcoholic fatty liver disease (NAFLD) is not well understood. We evaluated the molecular mechanisms that may be involved in the regulation of liver mtDNA content in a high-fat-induced rat model of NAFLD. In particular, we tested the hypothesis that liver mtDNA copy number is associated with liver expression of HIF-1α. Rats were given either standard chow diet (SCD, n = 10) or high-fat diet (HFD, n = 15) for 20 weeks. Subsequently, mtDNA quantification using nuclear DNA (nDNA) as a reference was carried out using real time quantitative PCR. HFD induced a significant increase in liver mtDNA/nDNA ratio, which significantly correlated with the liver triglyceride content (R: 0.29, P < 0.05). The liver mtDNA/nDNA ratio significantly correlated with the hepatic expression of HIF-1α mRNA (R: 0.37, P < 0.001); liver HIF-1α mRNA was significantly higher in the HFD group. In addition, liver cytochrome c oxidase subunit IV isoform 1 (COX4I1) mRNA expression was also positively correlated with liver mtDNA content. The hepatic expression of mRNA of transcriptional factors that regulate mitochondrial biogenesis, including peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and PGC-1ß, nuclear respiratory factor-1 (NRF-1), peroxisome proliferator-activated receptor δ and Tfam, was not associated with the liver mtDNA content. Neither hepatocyte apoptosis nor oxidative stress was involved in the HIF-1α-mediated increase in mtDNA copy number. In conclusion, we found that HFD promotes an increase in liver mitochondrial biogenesis in response to hypoxia via HIF-1α, probably to enhance the mitochondrial function as well as to accommodate the metabolic load.

Mitochondrial DNA synthesis studied autoradiographically in various cell types in vivo.

It is generally accepted that mitochondria are able to proliferate even in postmitotic cells due to their natural turnover and also to satisfy increased cell energy requirements. However, no detailed studies are available, particularly with respect to specific cell types. Since [3H]-thymidine is incorporated not only into nuclear (n) DNA but also into the DNA of cytoplasmic mitochondria, an autoradiographic approach was developed at the light microscopy level in order to study basic questions of mitochondrial (mt) proliferation in organs of rodents in situ via the cytoplasmic incorporation of [3H]-thymidine injected into the animals 1 h before sacrifice. Experiments carried out on mice after X-irradiation showed that cytoplasmic labeling was not due to a process such as unscheduled nuclear DNA synthesis (nUDS). Furthermore, half-lives of mitochondria between 8-23 days were deduced specifically in relation to cell types. The phase of mtDNA synthesis was about 75 min. Finally, mt proliferation was measured in brain cells of mice as a function of age. While all neurons showed a decreasing extent of mtDNA synthesis during old age, nUDS decreased only in distinct cell types of the cortex and hippocampus. We conclude that the leading theories explaining the phenomenon of aging are closely related, i.e., aging is due to a decreasing capacity of nDNA repair, which leads to unrepaired nDNA damage, or to an accumulation of mitochondria with damaged mtDNA, which leads to a deficit of cellular energy production.

ARS activity along the yeast mitochondrial apocytochrome b region: correlation with the location of petite genomes and consensus sequences.

Seven MboI fragments spanning the mitochondrial apocytochrome b gene in Saccharomyces cerevisiae strain D273-10B were cloned in the BamHI site of the integrative yeast vector YIp5 and the capacity for autonomous replication was subsequently assayed in yeast. The positive correlation found between the ars-like activity in four fragments and the presence of regions common to multiple ethidium bromide-induced petite (rho-) genomes suggests that the mitochondrial sequences possibly active as origins of replication in low-complexity neutral or weakly suppressive rho- mutants could be functionally related to the yeast nuclear replicator 11 nucleotide motif defined by Broach et al. (1983).