Nucleic Acids Research Advance Access published online on September 20, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm676
Molecular Biology |
The mitochondrial transcription termination factor mTERF modulates replication pausing in human mitochondrial DNA
1Institute of Medical Technology and Tampere University Hospital, FI-33014, University of Tampere, Finland, 2MRC-Dunn Human Nutrition Unit, Cambridge, UK, 3Department of Forensic Medicine and Tampere University Hospital, FI-33014, University of Tampere, Finland and 4Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, UK
*To whom correspondence should be addressed. Tel: +358 3 3551 7731; Fax: +358 3 3551 7710; E-mail: howard.t.jacobs{at}uta.fi
Received May 10, 2007. Revised August 8, 2007. Accepted August 19, 2007.
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.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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