共查询到20条相似文献,搜索用时 15 毫秒
1.
Samantha C. Lewis Priit Joers Smaranda Willcox Jack D. Griffith Howard T. Jacobs Bradley C. Hyman 《PLoS genetics》2015,11(2)
Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans. 相似文献
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Sulfolobus islandicus rod shaped virus 2 (SIRV2) infects the archaeon Sulfolobus islandicus at extreme temperature (70°C-80°C) and acidity (pH 3). SIRV2 encodes a Holliday junction resolving enzyme (SIRV2 Hjr) that has been proposed as a key enzyme in SIRV2 genome replication. The molecular mechanism for SIRV2 Hjr four-way junction cleavage bias, minimal requirements for four-way junction cleavage, and substrate specificity were determined. SIRV2 Hjr cleaves four-way DNA junctions with a preference for cleavage of exchange strand pairs, in contrast to host-derived resolving enzymes, suggesting fundamental differences in substrate recognition and cleavage among closely related Sulfolobus resolving enzymes. Unlike other viral resolving enzymes, such as T4 endonuclease VII or T7 endonuclease I, that cleave branched DNA replication intermediates, SIRV2 Hjr cleavage is specific to four-way DNA junctions and inactive on other branched DNA molecules. In addition, a specific interaction was detected between SIRV2 Hjr and the SIRV2 virion body coat protein (SIRV2gp26). Based on this observation, a model is proposed linking SIRV2 Hjr genome resolution to viral particle assembly. 相似文献
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Wild-type yeast mitochondrial DNA (mtDNA) is inherited biparentally, whereas mtDNA of hypersuppressive petite mutants is inherited uniparentally in crosses to strains with wild-type mtDNA. Genomes of hypersuppressive petites contain a conserved ori sequence that includes a promoter, but it is unclear whether the ori confers a segregation or replication advantage. Fluorescent in situ hybridization analysis of wild-type and petite mtDNAs in crosses reveals no preferential segregation of hypersuppressive petite mtDNA to first zygotic buds. We identify single-stranded DNA circles and RNA-primed DNA replication intermediates in hypersuppressive petite mtDNA that are absent from non-hypersuppressive petites. Mutating the promoter blocks hypersuppressiveness in crosses to wild-type strains and eliminates the distinctive replication intermediates. We propose that promoter-dependent RNA-primed replication accounts for the uniparental inheritance of hypersuppressive petite mtDNA. 相似文献
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Pathological mitochondrial DNA (mtDNA) rearrangements have been proposed to result from repair of double-strand breaks caused by blockage of mitochondrial DNA (mtDNA) replication. As mtDNA deletions are seen only in post-mitotic tissues, it has been suggested that they are selected out in actively dividing cells. By electron microscopy we observed rearranged mtDNA molecules in cultured human cells expressing a catalytically impaired helicase. As these molecules were undetectable by PCR, we propose that deleted mtDNA molecules in cultured cells are fragile and sensitive to heating. Further consequences of mtDNA replication stalling are discussed. 相似文献
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Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the mitochondrial replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation. 相似文献
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Joachim M. Gerhold Tiina Sedman Katarina Visacka Judita Slezakova Lubomir Tomaska Jozef Nosek Juhan Sedman 《The Journal of biological chemistry》2014,289(33):22659-22670
Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations. 相似文献
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Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes 总被引:1,自引:0,他引:1
Homologous recombination is required for maintaining genomic integrity by functioning in high-fidelity repair of DNA double-strand breaks and other complex lesions, replication fork support, and meiotic chromosome segregation. Joint DNA molecules are key intermediates in recombination and their differential processing determines whether the genetic outcome is a crossover or non-crossover event. The Holliday model of recombination highlights the resolution of four-way DNA joint molecules, termed Holliday junctions, and the bacterial Holliday junction resolvase RuvC set the paradigm for the mechanism of crossover formation. In eukaryotes, much effort has been invested in identifying the eukaryotic equivalent of bacterial RuvC, leading to the discovery of a number of DNA endonucleases, including Mus81?CMms4/EME1, Slx1?CSlx4/BTBD12/MUS312, XPF?CERCC1, and Yen1/GEN1. These nucleases exert different selectivity for various DNA joint molecules, including Holliday junctions. Their mutant phenotypes and distinct species-specific characteristics expose a surprisingly complex system of joint molecule processing. In an attempt to reconcile the biochemical and genetic data, we propose that nicked junctions constitute important in vivo recombination intermediates whose processing determines the efficiency and outcome (crossover/non-crossover) of homologous recombination. 相似文献
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Biased incorporation of ribonucleotides on the mitochondrial L-strand accounts for apparent strand-asymmetric DNA replication 总被引:15,自引:0,他引:15
Yang MY Bowmaker M Reyes A Vergani L Angeli P Gringeri E Jacobs HT Holt IJ 《Cell》2002,111(4):495-505
Recently, we presented evidence for conventional, strand-coupled replication of mammalian mitochondrial DNA. Partially single-stranded replication intermediates detected in the same DNA preparations were assumed to derive from the previously described, strand-asymmetric mode of mitochondrial DNA replication. Here, we show that bona fide replication intermediates from highly purified mitochondria are essentially duplex throughout their length, but contain widespread regions of RNA:DNA hybrid, as a result of the incorporation of ribonucleotides on the light strand which are subsequently converted to DNA. Ribonucleotide-rich regions can be degraded to generate partially single-stranded molecules by RNase H treatment in vitro or during DNA extraction from crude mitochondria. Mammalian mitochondrial DNA replication thus proceeds mainly, or exclusively, by a strand-coupled mechanism. 相似文献
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线粒体DNA复制及其调控 总被引:1,自引:0,他引:1
从线粒体DNA复制的模型与机制、复制的调控、复制忠实性及其损伤修复3个方面对近年来的研究文献进行了总结.在复制的模型与机制方面,对传统的D环复制的细节有了更深入的了解,新的实验方法的结果显示,在哺乳动物中还存在着链结合单向复制和链结合双向复制2种模型.在线粒体DNA复制的调控方面,近年来研究较多的调控因子主要包括mtDNA聚合酶γ、线粒体单链结合蛋白(mtSSB)、引物酶、解旋酶、连接酶、拓扑异构酶、转录因子mtTFA等,介绍了这些因子的最新研究进展及调控机制;对mtDNA复制时期和拷贝数量调控机制的研究也有突破,确定了Abf2p是mtDNA复制时期与拷贝数目的调控因子.在mtDNA复制的忠实性及其损伤修复研究方面,主要涉及到DNA Polγ的校正功能、错配修复、重组修复、DNA切除修复等,在mtDNA损伤修复中仅存在碱基切除修复机制,缺少核苷酸切除修复机制. 相似文献
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J. Müller‐Höcker R. Horvath S. Schäfer H. Hessel W. Müller‐Felber J. Kühr W. C. Copeland P. Seibel 《Journal of cellular and molecular medicine》2011,15(2):445-456
Combined morphological, immunocytochemical, biochemical and molecular genetic studies were performed on skeletal muscle, heart muscle and liver tissue of a 16‐months boy with fatal liver failure. The pathological characterization of the tissues revealed a severe depletion of mtDNA (mitochondrial DNA) that was most pronounced in liver, followed by a less severe, but still significant depletion in skeletal muscle and the heart. The primary cause of the disease was linked to compound heterozygous mutations in the polymerase γ (POLG) gene (DNA polymerase γ; A467T, K1191N). We present evidence, that compound heterozygous POLG mutations lead to tissue selective impairment of mtDNA replication and thus to a mosaic defect pattern even in the severely affected liver. A variable defect pattern was found in liver, muscle and heart tissue as revealed by biochemical, cytochemical, immunocytochemical and in situ hybridization analysis. Functionally, a severe deficiency of cytochrome‐c‐oxidase (cox) activity was seen in the liver. Although mtDNA depletion was detected in heart and skeletal muscle, there was no cox deficiency in these tissues. Depletion of mtDNA and microdissection of cox‐positive or negative areas correlated with the histological pattern in the liver. Interestingly, the mosaic pattern detected for cox‐activity and mtDNA copy number fully aligned with the immunohistologically revealed defect pattern using Pol γ, mtSSB‐ and mtTFA‐antibodies, thus substantiating the hypothesis that nuclear encoded proteins located within mitochondria become unstable and are degraded when they are not actively bound to mtDNA. Their disappearance could also aggravate the mtDNA depletion and contribute to the non‐homogenous defect pattern. 相似文献
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Medha Manchekar Karyn D. Scissum-Gunn Lisa A. Hammett Steffen Backert Brent L. Nielsen 《Plant science》2009,177(6):629-635
The structure of the mitochondrial genome in plants is unclear, but appears to consist of mostly linear DNA with some other structures, including branched molecules and subgenomic circles. Mitochondrial DNA (mtDNA) recombination was analyzed in Brassica campestris, which has one of the smallest mitochondrial genomes (218 kb) in higher plants. Field-inversion gel electrophoresis (FIGE) separated mtDNA into discrete populations that each represents the entire genome. Electron microscopy revealed large, mostly linear molecules trapped in the wells, slower migrating populations with mostly linear DNA and a low level of circular and networked mtDNA molecules of 10–140 kbp, and a fast migrating population of 10–50 kbp linear mtDNA. Some smaller than genome size circular molecules and circles with tails were observed, and may represent recombination or rolling circle replication intermediates. Hybridization of end-labeled mtDNA suggests there may be specific ends (or recombination hotspots) for some linear molecules. Analysis of mtDNA enriched by BND-cellulose and separated by two-dimensional agarose gel electrophoresis shows the presence of complex recombination structures and the presence of significant single-stranded regions in mtDNA. These findings provide further evidence that DNA recombination contributes to the complex structure of mtDNA in plants. 相似文献
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Li S 《DNA Repair》2011,10(1):65-72
Base excision repair (BER) of dimethyl sulfate induced N-methylpurines (NMPs) was measured at nucleotide resolution in the mitochondrial DNA (mtDNA) of cultured human and yeast (Saccharomyces cerevisiae) cells. NMPs were repaired with heterogeneous rates in the human mtDNA. The nearest-neighbor nucleotides significantly affected the repair rates: NMPs between pyrimidines were repaired much faster than those between purines, and those between a purine and a pyrimidine were repaired at intermediate rates. Repair intermediates of NMPs can also be detected at certain sites of the human mtDNA, indicating an ineffectiveness of processing the intermediates at these sites by the human mitochondrial BER machinery. In contrast to the human mtDNA, the yeast mtDNA did not show detectable repair of NMPs at any sites. Furthermore, a high level of spontaneous strand breaks exists exclusively at purine sites in the yeast mtDNA. Spontaneous NMPs or oxidative lesions were unlikely to be the major causes for the spontaneous strand breaks. Rather, spontaneous depurination combined with inefficient processing of DNA nicks or single-nucleotide gaps by the yeast mitochondrial BER machinery may result in the spontaneous strand breaks. Our results unveil a striking difference in BER between human and the yeast mitochondria. 相似文献
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The field of mitochondrial DNA (mtDNA) replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s) used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark) has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading- and lagging-strand synthesis (resembling bacterial genome replication) and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS). The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase γ, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase). Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells. 相似文献
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Mitotic homologous recombination is utilised to repair DNA breaks using either sister chromatids or homologous chromosomes as templates. Because sister chromatids are identical, exchanges between sister chromatids have no consequences for the maintenance of genomic integrity unless they involve repetitive DNA sequences. Conversely, homologous chromosomes might differ in genetic content, and exchanges between homologues might lead to loss of heterozygosity and subsequent inactivation of functional genes. Genomic instability, caused by unscheduled recombination events between homologous chromosomes, is enhanced in the absence of RecQ DNA helicases, as observed in Bloom's cancer-prone syndrome. Here, we used two-dimensional gel electrophoresis to analyse budding yeast diploid cells that were modified to distinguish replication intermediates originating from each homologous chromosome. Therefore, these cells were suitable for analysing the formation of inter-homologue junctions. We found that Rad51-dependent DNA structures resembling inter-homologue junctions accumulate together with sister chromatid junctions at damaged DNA replication forks in recQ mutants, but not in the absence of Srs2 or Mph1 DNA recombination helicases. Inter-homologue joint molecules in recQ mutants are less abundant than sister chromatid junctions, but they accumulate with similar kinetics after origin firing under conditions of DNA damage. We propose that unscheduled accumulation of inter-homologue junctions during DNA replication might account for allelic recombination defects in recQ mutants. 相似文献