The Experts below are selected from a list of 261 Experts worldwide ranked by ideXlab platform
David A. Clayton - One of the best experts on this subject based on the ideXlab platform.
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mitochondrial dna maintenance in vertebrates
Annual Review of Biochemistry, 1997Co-Authors: Gerald S. Shadel, David A. ClaytonAbstract:The discovery that mutations in mitochondrial DNA (mtDNA) can be pathogenic in humans has increased interest in understanding mtDNA maintenance. The functional state of mtDNA requires a great number of factors for gene expression, DNA replication, and DNA repair. These processes are ultimately controlled by the cell nucleus, because the requisite proteins are all encoded by nuclear genes and imported into the mitochondrion. DNA replication and transcription are linked in vertebrate mitochondria because RNA transcripts initiated at the light-Strand promoter are the primers for mtDNA replication at the Heavy-Strand origin. Study of this transcription-primed DNA replication mechanism has led to isolation of key factors involved in mtDNA replication and transcription and to elucidation of unique nucleic acid structures formed at this origin. Because features of a transcription-primed mechanism appear to be conserved in vertebrates, a general model for initiation of vertebrate Heavy-Strand DNA synthesis is proposed. In many organisms, mtDNA maintenance requires not only faithful mtDNA replication, but also mtDNA repair and recombination. The extent to which these latter two processes are involved in mtDNA maintenance in vertebrates is also appraised.
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rna dna hybrid formation at the human mitochondrial Heavy Strand origin ceases at replication start sites an implication for rna dna hybrids serving as primers
The EMBO Journal, 1996Co-Authors: Baoji Xu, David A. ClaytonAbstract:Abstract Critical elements of a mammalian mitochondrial DNA Heavy-Strand replication origin include a promoter and three downstream conserved sequence blocks (CSBIII, CSBII and CSBI). We found recently that a stable and persistent RNA-DNA hybrid forms during in vitro transcription at Saccharomyces cerevisiae mitochondrial origins; hybrid formation was dependent on the conserved CSBII element. We report here that during in vitro transcription with human mitochondrial RNA polymerase, stable and persistent RNA-DNA hybrid formation is also evident at the human mitochondrial Heavy-Strand origin. As predicted, hybrid formation was dependent on the GC-rich CSBII element. The human RNA-DNA hybrids terminate within or downstream of CSBI at locations implicated in initiation of mitochondrial DNA replication. Interestingly, efficient hybrid formation in the human system is influenced by sequence 5' to the RNA-DNA hybrid, including the CSBIII element. These results suggest that the RNA-DNA hybrids formed during transcription across the mitochondrial DNA Heavy-Strand origin provide RNA primers for initiation of mitochondrial DNA replication.
Craig E. Cameron - One of the best experts on this subject based on the ideXlab platform.
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New paradigms for regulation of human mitochondrial transcription (94.2)
The FASEB Journal, 2014Co-Authors: Craig E. CameronAbstract:Transcription of human mitochondrial DNA (mtDNA) is directed by three promoters: light-Strand promoter (LSP); Heavy-Strand promoter 1 (HSP1) and HSP2. Initiation from all promoters requires the mit...
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Transcription from the second Heavy-Strand promoter of human mtDNA is repressed by transcription factor A in vitro
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Maria F. Lodeiro, Akira Uchida, Megan Bestwick, Ibrahim M. Moustafa, Jamie J. Arnold, Gerald S. Shadel, Craig E. CameronAbstract:Cell-based studies support the existence of two promoters on the Heavy Strand of mtDNA: Heavy-Strand promoter 1 (HSP1) and HSP2. However, transcription from HSP2 has been reported only once in a cell-free system, and never when recombinant proteins have been used. Here, we document transcription from HSP2 using an in vitro system of defined composition. An oligonucleotide template representing positions 596–685 of mtDNA was sufficient to observe transcription by the human mtRNA polymerase (POLRMT) that was absolutely dependent on mitochondrial transcription factor B2 (TFB2M). POLRMT/TFB2M-dependent transcription was inhibited by concentrations of mitochondrial transcription factor A (TFAM) stoichiometric with the transcription template, a condition that activates transcription from the light-Strand promoter (LSP) in vitro. Domains of TFAM required for LSP activation were also required for HSP2 repression, whereas other mtDNA binding proteins failed to alter transcriptional output. Binding sites for TFAM were located on both sides of the start site of transcription from HSP2, suggesting that TFAM binding interferes with POLRMT and/or TFB2M binding. Consistent with a competitive binding model for TFAM repression of HSP2, the impact of TFAM concentration on HSP2 transcription was diminished by elevating the POLRMT and TFB2M concentrations. In the context of our previous studies of LSP and HSP1, it is now clear that three promoters exist in human mtDNA. Each promoter has a unique requirement for and/or response to the level of TFAM present, thus implying far greater complexity in the regulation of mammalian mitochondrial transcription than recognized to date.
Neal Sondheimer - One of the best experts on this subject based on the ideXlab platform.
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Topological requirements of the mitochondrial Heavy-Strand promoters.
Transcription, 2017Co-Authors: Ornella Zollo, Neal SondheimerAbstract:In vitro studies of mitochondrial transcription often use linear templates that fail to replicate key features of transcription on a circular genome. We developed a plasmid-based system for the analysis of Heavy-Strand promoters that recapitulates key features of native mtDNA to study topological and protein requirements of promoter activation. The Heavy-Strand promoters (HSP1 and HSP2) are simultaneously active on a circular template. HSP2 requires supercoiling for maximal activation. Increasing TFAM concentrations suppress HSP2 at levels that result in HSP1 stimulation. This study shows distinct modes of promoter activation, providing opportunities for the regulation of mitochondrial gene expression by promoter selection.
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Transcriptional requirements of the distal Heavy-Strand promoter of mtDNA
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Ornella Zollo, Valeria Tiranti, Neal SondheimerAbstract:The Heavy Strand of mtDNA contains two promoters with nonoverlapping functions. The role of the minor Heavy-Strand promoter (HSP2) is controversial, because the promoter has been difficult to activate in an in vitro system. We have isolated HSP2 by excluding its interaction with the more powerful HSP1 promoter, and we find that it is transcribed efficiently by recombinant mtRNA polymerase and mitochondrial transcription factor B2. The mitochondrial transcription factor A is not required for initiation, but it has the ability to alternatively activate and repress the HSP2 transcriptional unit depending on the ratio between mitochondrial transcription factor A and other transcription factors. The positioning of transcriptional initiation agrees with our current understanding of HSP2 activity in vivo. Serial deletion of HSP2 shows that only proximal sequences are required. Several mutations, including the disruption of a polycytosine track upstream of the HSP2 initiation site, influence transcriptional activity. Transcription from HSP2 is also observed when HeLa cell mitochondrial extract is used as the source of mitochondrial polymerase, and this transcription is maintained when HSP2 is provided in proper spacing and context to the HSP1 promoter. Studies of the linked Heavy-Strand promoters show that they are differentially regulated by ATP dosage. We conclude that HSP2 is transcribed and has features that allow it to regulate mitochondrial mRNA synthesis.
Gerald S. Shadel - One of the best experts on this subject based on the ideXlab platform.
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Transcription from the second Heavy-Strand promoter of human mtDNA is repressed by transcription factor A in vitro
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Maria F. Lodeiro, Akira Uchida, Megan Bestwick, Ibrahim M. Moustafa, Jamie J. Arnold, Gerald S. Shadel, Craig E. CameronAbstract:Cell-based studies support the existence of two promoters on the Heavy Strand of mtDNA: Heavy-Strand promoter 1 (HSP1) and HSP2. However, transcription from HSP2 has been reported only once in a cell-free system, and never when recombinant proteins have been used. Here, we document transcription from HSP2 using an in vitro system of defined composition. An oligonucleotide template representing positions 596–685 of mtDNA was sufficient to observe transcription by the human mtRNA polymerase (POLRMT) that was absolutely dependent on mitochondrial transcription factor B2 (TFB2M). POLRMT/TFB2M-dependent transcription was inhibited by concentrations of mitochondrial transcription factor A (TFAM) stoichiometric with the transcription template, a condition that activates transcription from the light-Strand promoter (LSP) in vitro. Domains of TFAM required for LSP activation were also required for HSP2 repression, whereas other mtDNA binding proteins failed to alter transcriptional output. Binding sites for TFAM were located on both sides of the start site of transcription from HSP2, suggesting that TFAM binding interferes with POLRMT and/or TFB2M binding. Consistent with a competitive binding model for TFAM repression of HSP2, the impact of TFAM concentration on HSP2 transcription was diminished by elevating the POLRMT and TFB2M concentrations. In the context of our previous studies of LSP and HSP1, it is now clear that three promoters exist in human mtDNA. Each promoter has a unique requirement for and/or response to the level of TFAM present, thus implying far greater complexity in the regulation of mammalian mitochondrial transcription than recognized to date.
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mitochondrial dna maintenance in vertebrates
Annual Review of Biochemistry, 1997Co-Authors: Gerald S. Shadel, David A. ClaytonAbstract:The discovery that mutations in mitochondrial DNA (mtDNA) can be pathogenic in humans has increased interest in understanding mtDNA maintenance. The functional state of mtDNA requires a great number of factors for gene expression, DNA replication, and DNA repair. These processes are ultimately controlled by the cell nucleus, because the requisite proteins are all encoded by nuclear genes and imported into the mitochondrion. DNA replication and transcription are linked in vertebrate mitochondria because RNA transcripts initiated at the light-Strand promoter are the primers for mtDNA replication at the Heavy-Strand origin. Study of this transcription-primed DNA replication mechanism has led to isolation of key factors involved in mtDNA replication and transcription and to elucidation of unique nucleic acid structures formed at this origin. Because features of a transcription-primed mechanism appear to be conserved in vertebrates, a general model for initiation of vertebrate Heavy-Strand DNA synthesis is proposed. In many organisms, mtDNA maintenance requires not only faithful mtDNA replication, but also mtDNA repair and recombination. The extent to which these latter two processes are involved in mtDNA maintenance in vertebrates is also appraised.
Maria Falkenberg - One of the best experts on this subject based on the ideXlab platform.
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Regulation of DNA replication at the end of the mitochondrial D-loop involves the helicase TWINKLE and a conserved sequence element
Nucleic Acids Research, 2015Co-Authors: Elisabeth Jemt, Örjan Persson, Marcela Dávila López, Christoph Freyer, Jay P Uhler, Majda Mehmedovic, Tore Samuelsson, Claes M Gustafsson, Maria FalkenbergAbstract:The majority of mitochondrial DNA replication events are terminated prematurely. The nascent DNA remains stably associated with the template, forming a triple-Stranded displacement loop (D-loop) structure. However, the function of the D-loop region of the mitochondrial genome remains poorly understood. Using a comparative genomics approach we here identify two closely related 15 nt sequence motifs of the D-loop, strongly conserved among vertebrates. One motif is at the D-loop 5′-end and is part of the conserved sequence block 1 (CSB1). The other motif, here denoted coreTAS, is at the D-loop 3′-end. Both these sequences may prevent transcription across the D-loop region, since light and Heavy Strand transcription is terminated at CSB1 and coreTAS, respectively. Interestingly, the replication of the nascent D-loop Strand, occurring in a direction opposite to that of Heavy Strand transcription, is also terminated at coreTAS, suggesting that coreTAS is involved in termination of both transcription and replication. Finally, we demonstrate that the loading of the helicase TWINKLE at coreTAS is reversible, implying that this site is a crucial component of a switch between D-loop formation and full-length mitochondrial DNA replication.
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conserved sequence box ii directs transcription termination and primer formation in mitochondria
Journal of Biological Chemistry, 2006Co-Authors: Xuan Hoi Pham, Martina Gaspari, Géraldine Farge, Claes M Gustafsson, Maria FalkenbergAbstract:Abstract The human mitochondrial transcription machinery generates the RNA primers needed for initiation of Heavy Strand DNA synthesis. Most DNA replication events from the Heavy Strand origin are prematurely terminated, forming a persistent RNA-DNA hybrid, which remains annealed to the parental DNA Strand. This triple-Stranded structure is called the D-loop and encompasses the conserved sequence box II, a DNA element required for proper primer formation. We here use a purified recombinant mitochondrial transcription system and demonstrate that conserved sequence box II is a sequence-dependent transcription termination element in vitro. Transcription from the light Strand promoter is prematurely terminated at positions 300-282 in the mitochondrial genome, which coincide with the major RNA-DNA transition points in the D-loop of human mitochondria. Based on our findings, we propose a model for primer formation at the origin of Heavy Strand DNA replication.