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

Abstract

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.

Genetic aspects of mitochondrial genome evolution

“…As an alternative to the strand displacement model, a coupled mode of leading and lagging strand replication, called strand-coupled model, is discussed in the literature (Holt et al, 2000;Yang et al, 2002). Contrary to the orthodox model it is assumed that a replication bubble originates preferentially from an interval located near O H spanning approximately one quarter of the mitogenome (Bowmaker et al, 2003;Reyes et al, 2005).…”

Genesis and Wanderings: Origins and Migrations in Asymmetrically Replicating Mitochondrial DNA

“…In subsequent publications, Holt and coworkers extensively modified and promoted that model as the sole mechanism of mtDNA replication, rather than an additional option to the strand-displacement mode. 4,5 This is important, since the mode and regulation of mtDNA replication will impact other aspects of mitochondrial genetics given that mtDNA replication, transcription, repair, recombination, and segregation are all linked to varying degrees, just as in other genomes. We accepted the idea that there might be alternative mechanisms of mtDNA replication, and initiated a study to determine the relative frequency of the two proposed modes in mouse liver mitochondria.…”

Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria

“…Because RNA/DNA hybrids of mtDNA can be lost readily during isolation (16,24), we also carried out a blocked-site assay to screen for retained primers. In MEFs lacking Rnaseh1, the failure to remove the LSP-Ori-H primer creates a stretch of RNA/DNA hybrid in the mtDNA that is refractive to restriction digestion at a site (np 16,179), immediately downstream of LSP (np 16,190) (SI Appendix, Fig.…”