Biased Incorporation of Ribonucleotides on the Mitochondrial L-Strand Accounts for Apparent Strand-Asymmetric DNA Replication

Recently, we presented evidence for conventional, strand-coupled replication of mammalian mitochondrial DNA (Holt et al. , 2000). Partially single-stranded replication intermediates detected in the same DNA preparations were assumed to derive from the previously described, strand-asymmetric mode of...

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Bibliographic Details
Published in:Cell 2002-11, Vol.111 (4), p.495-505
Main Authors: Yang, Ming Yao, Bowmaker, Mark, Reyes, Aurelio, Vergani, Lodovica, Angeli, Paolo, Gringeri, Enrico, Jacobs, Howard T., Holt, Ian J.
Format: Article
Language:English
Subjects:
Animals
DNA Replication - physiology
DNA, Mitochondrial - physiology
DNA, Single-Stranded
DNA-Binding Proteins - metabolism
Mitochondria, Liver
Nucleic Acid Hybridization
Rats
Ribonuclease H - metabolism
Ribonucleotides - physiology
RNA
RNA, Mitochondrial
Single-Strand Specific DNA and RNA Endonucleases - metabolism
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Summary:Recently, we presented evidence for conventional, strand-coupled replication of mammalian mitochondrial DNA (Holt et al. , 2000). 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.
ISSN:0092-8674
1097-4172
DOI:10.1016/S0092-8674(02)01075-9