Comparative analysis of mitochondrial genomes in different species reveals the evolution of G-quadruplexes

G-quadruplexes (G4s) are noncanonical structures that can form in the genomes of a range of organisms and are known to play various roles in cellular function. G4s can also form in mitochondrial DNA (mtDNA) because of their high guanine content, and these G4s may play roles in regulating gene expression, DNA replication, and genome stability. However, little is known regarding the evolution and dissemination of G4s in mitochondria. Here we analyzed the potential G4-forming sequences in mtDNA of 16 species from various families and demonstrated that the heavy strand of mtDNA of higher-order organisms contained higher levels of G4 regions than that of lower-order organisms. Analysis of the codons in the light strand revealed enrichment of guanine/cytosine-rich regions in higher eukaryotes and of adenine/thymidine-rich regions in lower-order organisms. Our study showed the diversity of G4s in species ranging from lower to higher orders. In particular, mammals such as humans, chimpanzees, and monkeys display a greater number of G4s than lower-order organisms. These potentially play a role in a range of cellular functions and assist in the evolution of higher organisms.

Polymerase γ efficiently replicates through many natural template barriers but stalls at the HSP1 quadruplex

Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters dsDNA and unique physical barriers such as structured genes, G-quadruplexes, and other obstacles. In vitro experiments here provide evidence that the polymerase γ heterotrimer is well-adapted to efficiently synthesize DNA, despite the presence of many naturally occurring roadblocks. However, we identified a specific G-quadruplex–forming sequence at the heavy-strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication. Furthermore, this structured region of DNA corresponds to the break site for a large (3,895 bp) deletion observed in mitochondrial disease patients. The presence of this deletion in humans correlates with UV exposure, and we have found that efficiency of polymerase γ DNA synthesis is reduced after this quadruplex is exposed to UV in vitro.

Mitochondrial genome of Chinese grass shrimp, Palaemonetes sinensis and comparison with other Palaemoninae species

The mitogenome of Chinese grass shrimp, Palaemonetes sinensis, was determined through Illumina sequencing, and the basic characteristics and gene arrangement were analyzed. The mitogenome of P. sinensis was 15955 bp in length, consisting of 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes and one control region, with tightly packed. 33 of these genes were encoded on the heavy strand, and the remainders encoded on the light strand. The composition of P. sinensis mitogenome presented a strong A + T bias, which account for 66.7%. All PCGs were initiated by a canonical ATN codon, except nad5, which was initiated by GTG. The termination codons of the PCGs were TAA, TAG and T–. The secondary structures of 22 tRNAs of P. sinensis had the typical clover structure, except of trnS1 owing to the lack of dihydroxyuridine (DHU) arm. Gene order comparison of P. sinensis and previously-sequenced Palaemoninae revealed a unique translocation between trnT and trnP in Macrobrachium. The phylogenetic analyses showed that three Exopalaemon species formed a monophyletic group and then clustered with two Palaemon species and P. sinensis successively whereas Macrobrachium clustered with Palaemon capensis in the other clade.

DNA repair and mutagenesis in vertebrate mitochondria: evidence for the asymmetric DNA strand inheritance

A variety of endogenous and exogenous factors induce chemical and structural alterations to cellular DNA, as well as errors occurring throughout DNA synthesis. These DNA damages are cytotoxic, miscoding, or both, and are believed to be at the origin of cancer and other age related diseases. A human cell, in addition to nuclear DNA, contains thousands copies of mitochondrial DNA (mtDNA), a double-stranded, circular molecule of 16,569 bp. It was proposed that mtDNA is a critical target for reactive oxygen species (ROS), by-products of the oxidative phosphorylation (OXPHOS), generated in the organelle during aerobic respiration. Indeed, oxidative damage to mtDNA are more extensive and persistent as compared to that of nuclear DNA. Although, transversions are the hallmarks of mutations induced by ROS, paradoxically, the majority of mtDNA mutations that occurred during ageing and cancer are transitions. Furthermore, these mutations exhibit a striking strand orientation bias: T→C/G→A transitions preferentially occur on the Light strand, whereas C→T/A→G on the Heavy strand of mtDNA. Here, we propose that the majority of mtDNA progenies, created after multiple rounds of DNA replication, are derived from the Heavy strand only, due to asymmetric replication of the DNA strand anchored to inner membrane via D-loop structure.

Mitochondrial mutational spectrum in mammals is sensitive to cellular and organismal longevity by means of A>G transitions

Mutational spectrum of the mitochondrial genome (mtDNA) does not resemble any of the known mutational signatures of the nuclear genome and variation in mtDNA mutational spectra between different tissues and organisms is still incomprehensible. Since mitochondria is tightly involved in energy production, we expect that mtDNA mutational spectra can reflect the level of cellular aerobic metabolism, which varies in different tissues. Analyzing a collection of somatic mtDNA mutations from human cancers, de novo mtDNA germline mutations from the human mother-offspring pairs, as well as mtDNA substitutions in hundreds of mammalian species, we observed that the frequency of AH>GH (heavy strand notation) substitutions is positively correlated with cellular and organismal longevity. For example, epithelium, oocytes of young mothers and mice have decreased AH>GH frequencies. We propose that AH>GH is a marker of cellular and organismal age, which is driven by oxidative damage of the single-stranded mtDNA during replication.Graphical abstractwhy melanoma is similar to a mouse and glioblastome resembles an elephant?

Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter

Human mtDNA contains three promoters, suggesting a need for differential expression of the mitochondrial genome. Studies of mitochondrial transcription have used a reductionist approach, perhaps masking differential regulation. Here we evaluate transcription from light-strand (LSP) and heavy-strand (HSP1) promoters using templates that mimic their natural context. These studies reveal sequences upstream, hypervariable in the human population (HVR3), and downstream of the HSP1 transcription start site required for maximal yield. The carboxy-terminal tail of TFAM is essential for activation of HSP1 but not LSP. Images of the template obtained by atomic force microscopy show that TFAM creates loops in a discrete region, the formation of which correlates with activation of HSP1; looping is lost in tail-deleted TFAM. Identification of HVR3 as a transcriptional regulatory element may contribute to between-individual variability in mitochondrial gene expression. The unique requirement of HSP1 for the TFAM tail may enable its regulation by post-translational modifications.

Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter

Human mtDNA contains three promoters, suggesting a need for differential expression of the mitochondrial genome. Studies of mitochondrial transcription have used a reductionist approach, perhaps masking differential regulation. Here we evaluate transcription from light-strand (LSP) and heavy-strand (HSP1) promoters using templates that mimic their natural context. These studies reveal sequences upstream, hypervariable in the human population (HVR3), and downstream of the HSP1 transcription start site required for maximal yield. The carboxy-terminal tail of TFAM is essential for activation of HSP1 but not LSP. Images of the template obtained by atomic force microscopy show that TFAM creates loops in a discrete region, the formation of which correlates with activation of HSP1; looping is lost in tail-deleted TFAM. Identification of HVR3 as a transcriptional regulatory element may contribute to between-individual variability in mitochondrial gene expression. The unique requirement of HSP1 for the TFAM tail may enable its regulation by post-translational modifications.