In vivo methylation of mtDNA reveals the dynamics of protein–mtDNA interactions

To characterize the organization of mtDNA–protein complexes (known as nucleoids) in vivo, we have probed the mtDNA surface exposure using site-specific DNA methyltransferases targeted to the mitochondria. We have observed that DNA methyltransferases have different accessibility to different sites on...

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Bibliographic Details
Published in:Nucleic acids research 2009-11, Vol.37 (20), p.6701-6715
Main Authors: Rebelo, Adriana P., Williams, Sion L., Moraes, Carlos T.
Format: Article
Language:English
Subjects:
Binding Sites
Cell Line
Cell Nucleus - metabolism
DNA - metabolism
DNA Methylation
DNA Replication
DNA, Mitochondrial - biosynthesis
DNA, Mitochondrial - chemistry
DNA, Mitochondrial - metabolism
DNA-Binding Proteins - metabolism
Gene Regulation, Chromatin and Epigenetics
Methyltransferases - metabolism
Mitochondria - enzymology
Mitochondria - metabolism
Oxidative Stress
Transcription Factors - metabolism
Transcription, Genetic
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Summary:To characterize the organization of mtDNA–protein complexes (known as nucleoids) in vivo, we have probed the mtDNA surface exposure using site-specific DNA methyltransferases targeted to the mitochondria. We have observed that DNA methyltransferases have different accessibility to different sites on the mtDNA based on the levels of protein occupancy. We focused our studies on selected regions of mtDNA that are believed to be major regulatory regions involved in transcription and replication. The transcription termination region (TERM) within the tRNALeu(UUR) gene was consistently and strongly protected from methylation, suggesting frequent and high affinity binding of mitochondrial transcription termination factor 1 (mTERF1) to the site. Protection from methylation was also observed in other regions of the mtDNA, including the light and heavy strand promoters (LSP, HSP) and the origin of replication of the light strand (OL). Manipulations aiming at increasing or decreasing the levels of the mitochondrial transcription factor A (TFAM) led to decreased in vivo methylation, whereas manipulations that stimulated mtDNA replication led to increased methylation. We also analyzed the effect of ATAD3 and oxidative stress in mtDNA exposure. Our data provide a map of human mtDNA accessibility and demonstrate that nucleoids are dynamically associated with proteins.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkp727