Yeast RAD51 gene functions in genetic recombination and DNA double-strand break repair. In vitro, in the presence of ATP and replication protein A, RAD51 protein pairs single-stranded DNA (ssDNA) with homologous double-stranded DNA (dsDNA) and catalyzes strand exchange between the synapsed DNA partners. Electron microscopic analyses show that RAD51 forms helical filaments on both ssDNA and dsDNA, in which the DNA is highly extended. However, results presented here indicate that only the RAD51-ssDNA nucleoprotein filament is functionally relevant. Strand exchange is arrested when heterology is encountered in the duplex partner, and analysis of the configuration of the terminal joint thus formed reveals that pairing and strand exchange initiate at the 5' end of the complementary strand in the linear duplex, a reaction polarity opposite to that of the bacterial prototype RecA.

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)

Benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide (BPDE), a metabolite of the widespread environmental pollutant benzo[a]pyrene, is a mutagenic in both bacterial and mammalian systems. Toward understanding the mutagenic effects of different stereoisomers of BPDE at specific sites in DNA, six stereochemically defined BPDE adducts were constructed on adenine N6 at position 2 of the human N-ras 61 codon within an 11-base oligonucleotide fragment. Both the nonadducted and BPDE-adducted N-ras 61 11-mers were inserted into a unique EcoRI site in single-stranded M13mp7L2 DNA and utilized for in vivo studies. The ligation efficiencies of BPDE-adducted 11-mers into the single-stranded vector were determined by Southern hybridization and confirmed by electron microscopy. Repair-deficient AB2480 E. coli cells were transformed with adducted and non-adducted DNA samples. The resultant plaque-forming abilities were used to evaluate the replication competence of the various BPDE adducts with respect to the nonadducted 11-mer. Point mutations due to aberrant replication at the adducted site were identified by the technique of differential DNA hybridization. All of the six BPDE adducts examined were mutagenic in vivo, generating exclusively A-->G mutations at frequencies ranging from 0.26 to 1.20%. In vitro replication studies using these BPDE-adducted 11-mers involved primer extension assays with Klenow fragment. All of the BPDE-modified templates demonstrated distinct blockage at the adducted site and/or 1 base 3' to the adducted site, allowing essentially no translesion synthesis to form fully extended polymerization products in vitro.

The culture medium of Pseudomonas BAL 31 contains endonuclease activities which are highly specific for single-stranged DNA and for the single-stranded or weakly hydrogen-bonded regions in supercoiled closed circular DNA. Exposure of nicked DNA to the culture medium results in cleavage of the strang opposite the sites of preexisting single-strand scissions. At least some of the linear duplex molecules derived by cleavage of supercoiled closed circular molecules contain short single-stranded ends. Single-strand scissions are not introduced into intact, linear duplex DNA or unsupercoiled covalently closed circular DNA. Under these same reaction conditions, 0X174 phage DNA is extensively degraded and PM2 form I DNA is quantitatively converted to PM2 form III linear duplexes. Prolonged exposure of this linear duplex DNA to the concentrated culture medium reveals the presence of a double-strand exonuclease activity that progressively reduces the average length of the linear duplex. These nuclease activities persist at ionic strengths up to 4 M and are not eliminated in the presence of 5% sodium dodecyl sulfate. Calcium and magnesium ion are both required for optimal activity. Although the absence of magnesium ion reduces the activities, the absence of calcium ion irreversibly eliminates all the activities.

Thymidine kinase-lines of mouse L cells incorporate thymidine exclusively into mitochondrial DNA. This fact permits assessment of labeled. mitochondrial DNA components in ethidium bromide-cesium chloride gradients. Contaminating nuclear DNA is unlabeled and need not be removed. Elimination of the DNase treatment of purified mitochondria reveals that the replicative forms that exhibit displacement replication up to full genome size are covalently-closed circularmolecules. Denaturation followed by brief renaturation of these larger replicating molecules produces closed-circular DNA with a deficiency of Watson-Crick turns, appearing as singlestrand loops. This result suggests that displacement replication proceeds with nicking and rapid closure of the covalently-closed circular template.Thymidine kinase-lines of mouse L cells incorporate exogenous thymidine exclusively into the mitochondrial DNA (mtDNA) of the cells (1), and they contain thymidine kinase only in their mitochondria (Berk, A. J. & Clayton, D. A., submitted). These aspects of thymidine kinase-cells permit detection of thymidine-labeled mature mtDNA and replicative intermediates (2-4) in ethidium bromide-CsCl gradients in the presence of unlabeled nuclear DNA. MATERIALS AND METHODSCells. The thymidine kinase-counterpart of LA9, LMTK-, and the thymidine kinase-derivative of the circular dimer containing line LD, LDTK-, were isolated by growth in increasing amounts of BrdU (5). LMTK-has been used in earlier studies (1). The LDTK-line is introduced here. The cells were grown as described (1).Preparation of mtDNA. Cells labeled with [3H]thymidine (0.5 ,Ci/ml, 3 Ci/mmol) were harvested in log phase at densities of 3 to 7 X 105/ml. Stationary phase is reached under these growth conditions at 1.3 X 106 cells per ml. The cells were washed with 30 volumes of reticulocyte standard buffer (RSB) (10 mM NaCl-1.5 mM MgCl2-10 mM Tris-HCl, pH 7.5) and then suspended in RSB at a ratio of 20 ml/0.5 ml of wet packed unswollen cells. Swelling was allowed to continue for 5 min, after which the cells were broken by' 7-15 passages in a Dounce homogenizer. 2.0 M sucrose in RSB was then added to bring the final concentration to 0.28 M, and nuclei were removed by centrifugation at 2500 rpm in a Sorvall GLC-1 for 5 min. Mitochondria were then purified by one-step (3) or two-step (1) sucrose gradients, collected from the interface of these gradients, suspended to 1 ml in 0.5 M NaCl-50 mM Tris-10 mM EDTA, pH 8.5, and lysed by addition of 1 ml of 2% sodium dodecyl sulfate dissolved in the same buffer warmed to 37°. There is no apparent difference in results obtained from the one-step or two-step sucrose gradients. Incubation at 370 for 3 min completed the lysis. Solid CsCl was added to a final density of 1.55 g/ml, and ethidium bromide was added to a final concentration of 350 ,ug/ml. The samples were centrifuged in a polyallomer tube in an SW50.1 rotor for 18 hr at 38,000 rpm at 200, after which the contents of the tube were poured into a second polyallomer tube. The volum...

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The synthesis of a complementary DNA copy (cDNA) of hen ovalbumin mRNA using AMV RNA-directed DNA polymerase was studied under different conditions of salt, deoxyribonucleotide concentrations, temperature, and time. It was observed that in the absence of monovalent cation at 46 degrees C a complete transcript of ovalbumin mRNA could be effected by the enzyme. The minimum deoxyribonucleotide requirement for complete synthesis was 35 muM for dATP, dGTP, and dCTP and 200 muM dTTP. By a number of different experimental criteria which included sedimentation on alkaline sucrose gradients and electrophoresis in polyacrylamide gels containing 98% formamide, direct electron microscope visualization, and protection of ovalbumin [25I]mRNA from nuclease digestion it could be demonstrated that a considerable fraction of a complete mRNA transcript was indeed synthesized. The cDNA/ovalbumin mRNA hybrid had a Tm on hydroxylapatite of 92 degrees C, indicating the synthesis of a RNA transcript with a high fidelity. When such a complete ovalbumin [3H]cDNA was synthesized with a specific activity of 10(8) cpm/mug and hyfridized to an excess of chick DNA, the kinetics of hybridization indicated that the cDNA was comprised of a nonrepetitive sequence.