TY - JOUR
T1 - The mitochondrial transcription termination factor mTERF modulates replication pausing in human mitochondrial DNA
AU - Hyvärinen, Anne K.
AU - Pohjoismäki, Jaakko L.O.
AU - Reyes, Aurelio
AU - Wanrooij, Sjoerd
AU - Yasukawa, Takehiro
AU - Karhunen, Pekka J.
AU - Spelbrink, Johannes N.
AU - Holt, Ian J.
AU - Jacobs, Howard T.
N1 - Funding Information:
This work was supported by funding from the Academy of Finland, Tampere University Hospital Medical Research Fund, the UK Medical Research Council, the UK Muscular Dystrophy Campaign, the Juselius Foundation and the European Union EUMITOCOMBAT and MitEURO projects. We thank Steffi Goffart for advice and assistance with mtDNA copy number determination and interpretation of 2DNAGE, Peter Martinsson for help with microscopy, and Gertjan Hakkaart, Merja Jokela, Outi Kurronen, Anja Rovio, Tea Tuomela, Päivi Turunen and Ernesto Zanotto for advice and technical assistance. We also thank Claes Gustafsson, Heather Lorimer, Sanna Marjavaara, Maria Sola Villarubias and Anu Wartiovaara, as well as many colleagues in the MitEURO network, for useful discussions. Funding to pay the Open Access publication charges for this article was provided by University of Tampere and the Academy of Finland.
PY - 2007/10
Y1 - 2007/10
N2 - The mammalian mitochondrial transcription termination factor mTERF binds with high affinity to a site within the tRNALeu(UUR) gene and regulates the amount of read through transcription from the ribosomal DNA into the remaining genes of the major coding strand of mitochondrial DNA (mtDNA). Electrophoretic mobility shift assays (EMSA) and SELEX, using mitochondrial protein extracts from cells induced to overexpress mTERF, revealed novel, weaker mTERF-binding sites, clustered in several regions of mtDNA, notably in the major non-coding region (NCR). Such binding in vivo was supported by mtDNA immunoprecipitation. Two-dimensional neutral agarose gel electrophoresis (2DNAGE) and 5′ end mapping by ligation-mediated PCR (LM-PCR) identified the region of the canonical mTERF-binding site as a replication pause site. The strength of pausing was modulated by the expression level of mTERF. mTERF overexpression also affected replication pausing in other regions of the genome in which mTERF binding was found. These results indicate a role for TERF in mtDNA replication, in addition to its role in transcription. We suggest that mTERF could provide a system for coordinating the passage of replication and transcription complexes, analogous with replication pause-region binding proteins in other systems, whose main role is to safeguard the integrity of the genome whilst facilitating its efficient expression.
AB - The mammalian mitochondrial transcription termination factor mTERF binds with high affinity to a site within the tRNALeu(UUR) gene and regulates the amount of read through transcription from the ribosomal DNA into the remaining genes of the major coding strand of mitochondrial DNA (mtDNA). Electrophoretic mobility shift assays (EMSA) and SELEX, using mitochondrial protein extracts from cells induced to overexpress mTERF, revealed novel, weaker mTERF-binding sites, clustered in several regions of mtDNA, notably in the major non-coding region (NCR). Such binding in vivo was supported by mtDNA immunoprecipitation. Two-dimensional neutral agarose gel electrophoresis (2DNAGE) and 5′ end mapping by ligation-mediated PCR (LM-PCR) identified the region of the canonical mTERF-binding site as a replication pause site. The strength of pausing was modulated by the expression level of mTERF. mTERF overexpression also affected replication pausing in other regions of the genome in which mTERF binding was found. These results indicate a role for TERF in mtDNA replication, in addition to its role in transcription. We suggest that mTERF could provide a system for coordinating the passage of replication and transcription complexes, analogous with replication pause-region binding proteins in other systems, whose main role is to safeguard the integrity of the genome whilst facilitating its efficient expression.
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U2 - 10.1093/nar/gkm676
DO - 10.1093/nar/gkm676
M3 - Article
C2 - 17884915
AN - SCOPUS:36148945648
SN - 0305-1048
VL - 35
SP - 6458
EP - 6474
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 19
ER -