Replication of Mitochondrial DNA. Circular Replicative Intermediates in Mouse L Cells

Abstract

The frequency, composition, and structure of circular replicating forms of mitochondrial DNA, including two new forms described here, suggest a scheme for the mode of replication of this DNA.The closed-circular mitochondrial DNA (mtDNA) isolated from exponentially growing mouse L cells contains a substantial fraction of displacement-loop molecules (1). These molecules, formed by displacement synthesis in the first discrete stage of DNA replication, contain a short progeny strand hydrogen-bonded at a unique site to the light parental strand. This communication describes the structure of larger circular replicative intermediates that appear to have been formed upon the continuation of displacement synthesis and the subsequent asynchronous synthesis of light strand on the displaced heavy strand. A sequential arrangement of the replicative structures has suggested a scheme for certain aspects of the replication of mtDNA (Fig. 1). This scheme is given here so as to simplify the l)resentation of the results on which it is based. MATERIALS AND METHODSThe radioactive mtDNA and the separate mtDNA complements were prepared as described (1). The upper and lower bands from the final ethidium bromide-CsCl density gradient were fractionated as shown in Fig. 2 of ref. 1, except that the central four fractions, rich in catenanes, were not included in the upper-band sample. The hybridizations were performed at 70'C in 3 M CsCl, pH 8.5, for 4 hr.The electron-microscope experiments were performed as described (1)

Genetic aspects of mitochondrial genome evolution

“…The most widely accepted "orthodox" hypothesis explaining the mode of mtDNA replication is the so called strand displacement model (Robberson et al, 1972;Goddard and Wolstenholme, 1980;Clayton, 1982). We can give here only a brief outline and refer to the reviews by Shadel and Clayton (1997) and Taanman (1999) for much more detailed description of the replication process.…”

The Absence of a Pyrimidine Dimer Repair Mechanism in Mammalian Mitochondria

“…Thus, the most reasonable explanation for all these data is that mtDNA molecules that contain pyrimidine dimers are not repaired but replicate until a dimer is encountered on a parental strand and then replication ceases. If this occurs early in replication [before synthesis of 0.6 genome lengths (8)], the molecule will be arrested as an expanded D-loop molecule. If this occurs late in replication, then the daughter molecules can separate and are isolated as gapped circular molecules that cannot complete duplex synthesis and subsequent closure.…”

“…We were therefore interested in determining whether or not mammalian cells contain a repair mechanism(s) for removal of ultraviolet light-induced pyrimidine dimers in mitochondrial DNA (mtDNA). In addition, recent work has established that these DNA molecules replicate in situ and the mechanism of replication has been described (8)(9)(10)(11). If mammalian mitochondria could be shown to carry out repair of damaged DNA, it is possible that some of the previously described presumptive replicating forms of mtDNA might in part be repair intermediates.…”

Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro

“…DNA synthesis from O H is unidirectional and proceeds to displace the parental heavy strand (H-strand). After leading-strand synthesis has reached two-thirds of the genome, it activates O L and lagging-strand DNA synthesis then initiates in the opposite direction (2)(3)(4). Recently, analysis of mtDNA replication intermediates by atomic force microscopy revealed that the strand-displacement model is an oversimplification and that additional initiation sites for lagging-strand mtDNA synthesis exist on the light strand (L-strand) (5).…”