A Novel Closed-Circular Mitochondrial DNA with Properties of a Replicating Intermediate

Abstract

A fraction of the covalently closed mitochondrial DNA in mouse L cells contains a replicated heavy-strand segment that is hydrogen bonded to the circular light strand. The inserted single strand is dissociable from the circular duplex at an elevated temperature.M1itochondrial DNA from animal cells occurs in the form of closed-circular molecules with a molecular weight of about 10 X 100 (1). In the course of a study of the mechanism of replication of this DNA in two strains of mouse L cells, we have found that about one half of the covalently closed molecules in exponentially growing cells contain a short threestranded DNA region, which we have called a D-loop, or displacement loop (Fig. 1). The D-loop appears to have been formed in the closed DNA by displacement synthesis of a short progeny strand with a specific region of the mitochondrial light strand serving as a template. This communication presents the results of experiments designed to elucidate the -structure and properties of this new kind of closedcircular DNA and considers its role as an intermediate in the replication process. MATERIALS AND METHODSThe two cell lines studied were the LD line (our designation) obtained from C. Schildkraut, Albert Einstein College of Medicine, New York, and the LA9 line isolated by Littlefield (2) and obtained from L. Crawford, Imperial Cancer Research Fund, London. Mitochondrial DNA (mtDNA) in the LD cells occurs in the form of circular dimers with a contour length of 10 Am. The LA9 cells contain monomeric mtDNA.The spinner-adapted cells were grown in Dulbecco's modification of Eagle's phosphate medium, containing 10% calf serum. The generation times of the LA9 and LD cells were 22 and 20 hr, respectively. Exponentially growing cells, about 2 X 105 cells/ml, were labeled with 0.5 MCi/ml of [3H]thymidine (Schwarz/Mann), 15.6 Ci/mmol, for 3 hr before harvest.

Origin and direction of replication in mitochondrial DNA molecules from Drosophila melanogaster.

“…D loop-containing molecules have been found in mtDNA from many metazoans, and the D loop has been identified as the first step in replication of mouse L-cell mtDNA (18,19). Failure to find a distinct excess of D loop-containing D. melanogaster mtDNA molecules in this study and previously (5,7,25) may indicate a real difference between the mechanisms of replication operative in these organisms and other metazoan mtDNAs studied, 'as recently suggested by Rubenstein et al (25).…”

Association of a protein structure of probable membrane derivation with HeLa cell mitochondrial DNA near its origin of replication.

“…Evidence against an Artifactual Nature of the mtDNAProtein Complexes. To exclude the possibility that these complexes artifactually formed during lysis of mitochondria (possibly due to sticking of proteins or membrane fragments to the single-strand DNA portion of the D-loop), 32P-labeled closed-circular mtDNA [which contains the majority of the D-loop DNA (14,15)] was mixed with lysis buffer before addition to a [3H]thymidine-labeled mitochondrial suspension; one half of the lysate was treated with formaldehyde and glutaraldehyde, the other half was left untreated, and mtDNAprotein complexes were isolated from the two portions of the lysate. As shown in Fig.…”

Isolation and Partial Characterisation of the Relaxation Protein from Nuclei of Cultured Mouse and Human Cells

“…It is clear, however, that the relaxation protein can replace the combined successive actions of endonuclease and ligase in the development of transient swivels in duplex DNA. Such swivels are indispensable in the replication of closed-circular duplex DNA, which has been shown to form closed intermediates [21,22]. Such transient swivels could also provide the useful function of eliminating the need for the rapid rotation of the entire parental duplex ahead of the advancing replication fork in chromosomal DNA.…”