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.

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)

For nuclear entry of large nucleoprotein complexes, it is thought that one key nuclear localization signal (NLS) of a protein component becomes exposed to mediate importin recognition. We show that the nuclear entry of simian virus 40 involves a dynamic interplay between two distinct interiorly situated capsid NLSs, the Vp1 NLS and the Vp3 NLS, and the selective exposure and importin recognition of the Vp3 NLS. The Vp3 NLS-null mutants assembled normally into virion-like particles (VLP) in mutant DNA-transfected cells. When used to infect a new host, the null VLP entered the cell normally but was impaired in viral DNA nuclear entry due to a lack of recognition by the importin ␣2/␤ heterodimer, leading to reduced viability. Both Vp3 and Vp1 NLSs directed importin interaction in vitro, but the Vp1 NLS, which overlaps the Vp1 DNA binding domain, did not bind importins in the presence of DNA. The results suggest that certain canonical NLSs within a nucleoprotein complex, such as the Vp1 NLS, can be masked from functioning by binding to the nucleic acid component and that the availability of an NLS that is not masked and can become exposed for importin binding, such as the Vp3 NLS, is a general feature of the nuclear entry of the nucleoprotein complexes, including those of other animal viruses.

The covalent oligomer formation is blocked in the presence of a sulfhydryl-modifying reagent. We propose that there are two stages in this Vp1 disulfide bonding. First, the newly synthesized Vp1 monomers acquire intrachain bonds as they fold and begin to interact. Next, these bonds are replaced with intermolecular bonds as the monomers assemble into pentamers. This sequential appearance of transitory disulfide bonds is consistent with a role for sulfhydryldisulfide redox reactions in the coordinate folding of Vp1 chains into pentamers. The cytoplasmic Vp1 does not colocalize with marker proteins of the endoplasmic reticulum. This paper demonstrates in vivo disulfide formations and exchanges coupled to the folding and oligomerization of a mammalian protein in the cytoplasm, outside the secretory pathway. Such disulfide dynamics may be a general phenomenon for other cysteine-bearing mammalian proteins that fold in the cytoplasm. H ow proteins fold into functional, three-dimensional structures has been under intense study (1), and the folding pathways for a number of eukaryotic proteins have been characterized in vitro (2, 3) or in vivo (4-9). In the secretory pathway, protein folding is coupled to the formation and reshuffling of disulfide bonds. These redox conversions, leading to native, disulfide-bonded proteins, are catalyzed by prokaryotic Dsb proteins in the periplasm (10-12) and eukaryotic protein disulfide isomerase (PDI) in the endoplasmic reticulum (ER) (13-16). Proteins that fold and assemble in the reducing environment of the cytoplasm generally do not harbor native disulfides, owing to the activities of thioredoxins and glutaredoxins. Transitory disulfide bonding, though, is required for the folding of bacteriophage P22 tailspike protein in the cytoplasm (17, 18). Whether disulfide bond-coupled folding pathways exist for nonsecretory proteins in the mammalian cytoplasm is not known.The structure of simian virus 40 (SV40), known at the atomic resolution, is determined by the major capsid protein Vp1 (19). Seventy-two pentamers of Vp1 form the outer shell of SV40, with each monomer making contact with its four intrapentamer neighbors via interdigitating secondary structural elements. The Vp1 pentamer is expected to form in the cytoplasm of SV40-infected cells during or soon after the monomers' synthesis (20, 21). There are seven cysteine residues in one Vp1 chain. No intrapentamer disulfide bridges, either between or within the monomers, are observed in the mature particle (22). Certain cysteine residues do lie in close proximity of one another, such as the Cys-49-Cys-87 and Cys-87-Cys-207 pairs within one monomer and the Cys-49-Cys-207 pair between two monomers within a pentamer (19). Each cysteine pair conceivably can become juxtaposed during the folding process and form a transient disulfide bond.In this study, we show that in the virus-infected cytoplasm, the newly synthesized Vp1 chain is an intramolecularly disulfidebonded monomer and is a precursor for intermolecularly disulfidebonded Vp1 oligome...

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