Using two-dimensional agarose gel electrophoresis, we show that mitochondrial DNA (mtDNA) replication of birds and mammals frequently entails ribonucleotide incorporation throughout the lagging strand (RITOLS). Based on a combination of two-dimensional agarose gel electrophoretic analysis and mapping of 5' ends of DNA, initiation of RITOLS replication occurs in the major non-coding region of vertebrate mtDNA and is effectively unidirectional. In some cases, conversion of nascent RNA strands to DNA starts at defined loci, the most prominent of which maps, in mammalian mtDNA, in the vicinity of the site known as the light-strand origin.

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.

Previous data from our laboratory suggested that replication of mammalian mitochondrial DNA initiates exclusively at or near to the formerly designated origin of heavy strand replication, O H , and proceeds unidirectionally from that locus. New results obtained using twodimensional agarose gel electrophoresis of replication intermediates demonstrate that replication of mitochondrial DNA initiates from multiple origins across a broad zone. After fork arrest near O H , replication is restricted to one direction only. The initiation zone of bidirectional replication includes the genes for cytochrome b and NADH dehydrogenase subunits 5 and 6.