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. 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.
Yang
Ming Yao
MY
MRC-Dunn Human Nutrition Unit, Hills Road, CB2 2XY, Cambridge, United Kingdom.
Bowmaker
Mark
M
Reyes
Aurelio
A
Vergani
Lodovica
L
Angeli
Paolo
P
Gringeri
Enrico
E
Jacobs
Howard T
HT
Holt
Ian J
IJ
D000818:Animals; D004261:DNA Replication / Q000502:physiology*; D004272:DNA, Mitochondrial / Q000502:physiology*; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000378:metabolism; D008930:Mitochondria, Liver; D009693:Nucleic Acid Hybridization; D012313:RNA; D000077278:RNA, Mitochondrial; D051381:Rats; D016914:Ribonuclease H / Q000378:metabolism; D012265:Ribonucleotides / Q000502:physiology; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D000077278:RNA, Mitochondrial; D012265:Ribonucleotides; D012313:RNA; D016914:Ribonuclease H; D015719:Single-Strand Specific DNA and RNA Endonucleases