Summary
When Bacillus subtilis infected with virus 2C was labeled with short pulses of tritiated uracil (a specific precursor of the hydroxymethyluracil replacing thymine in 2C chromosome) and the cell lysate was fractionated in alkaline sucrose gradients, most of the radioactivity was found within a lowmolecular-weight peak of DNA (“Okazaki pieces”) of about 2×106 daltons. After longer pulses, segments of large size and full strands were the main labeled products. A shift from low to high-molecular-weight nascent DNA took place slowly during the eclipse phase, and rapidly during the maturation phase. This suggested a lagging of the ligase behind the polymerase at early stages. During the entire replication cycle, pulse-labeled DNA was present within the replication fork in its native form with no involvement of proteins. Furthermore, the amount of lowmolecular-weight pulse-labeled DNA hybridizing with the L strand was higher than that annealing with the H strand. To explain this finding it is suggested that the polymerization or the ligation of small segments is faster for one strand than for the other. Finally, the DNA fraction which is resistant to single-strand specific nuclease was about 10% when alkali-denatured Okazaki pieces were neutralized at 4C and was increased about five-fold upon selfannealing. It is concluded that both strands of 2C DNA duplicate in a discontinuous fashion through the formation of Okazaki pieces, which are held by hydrogen bonds initially and subsequently by phosphodiester linkages. It is proposed also that ligase action is faster for the L strand than for the H strand, and that this phenomenon might be related to both the polarity of chain growth and the asymmetry of transcription of 2C chromosome.
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References
Andersen, J.J., Ganesan, A.T.: DNA synthesis in vivo in B. subtilis. J. Mol. Biol. 106, 285–295 (1976)
Bernardi, G.: Chromatography of nucleic acids on hydroxyapaptite columns. pp. 455–500. In: Procedures in nucleic acid research. G.L. Cantoni and D.R. Davies (ed.) vol 2. New York: Harper and Rowe 1971
Burgi, E., Hershey, A.D.: Sedimentation rate as a measure of molecular weight of DNA. Biophys. J. 36, 309–321 (1963)
Center, M.S.: Bacteriophage T7 DNA synthesis in isolated DNA-membrane complexes. J. Virol. 12, 847–854 (1973)
Cocito, C.: The action of virginiamycin on nucleic acid and protein synthesis in B. subtilis infected with bacteriophage 2C. J. Gen. Microbiol. 57, 195–206 (1969)
Cocito, C.: Origin and metabolic properties of the RNA species formed during the replication cycle of virus 2C. J. Virol. 14, 1482–1493 (1974)
Delius, H., Howe, C., Kozinski, A.W.: Structure of the replicating DNA from bacteriophage T4. Proc. Natl. Acad. Sci. U.S.A. 68, 3049–3053 (1971)
Denhardt, D.T.: A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641–646 (1966)
Dressler, D.: The recent excitement in the DNA growing point problem. Annu. Rev. Microbiol. 29, 525–559 (1975)
Fareed, G.C., Garon, C.F., Salzmann, N.P.: Origin and direction of simian virus 40 deoxyribonucleic acid replication. J. Virol. 10, 484–491 (1972)
Fareed, G.C., Khoury, G., Salzmann, N.P.: Self-annealing of 4S strands from replicating simian virus 40 DNA. J. Mol. Biol. 77, 457–562 (1973)
Francke, B., Hunter, T.: In vitro polymoma DNA synthesis: discontinuous chain growth. J. Mol. Biol. 83, 99–121 (1974)
Gefter, M.L.: DNA replication. Annu. Rev. Biochem 44, 45–78 (1975)
Ginsberg, B., Hurwitz, J.: Unbiased synthesis of pulse-labeled DNA fragments of bacteriophage λ and T4. J. Mol. Biol. 52, 265–280 (1970)
Hoet, P., Coene, M., Cocito, C.: Synthesis of phage 2C-DNA in permeabilized B. subtilis. Mol. Gen. Genet. 158, 297–303 (1978)
Hoet, P., Fraselle, G., Cocito, C.: Transfer to progeny of both DNA strands of phage 2C. Biochem. Biophys. Res. Commun. 66, 235–241 (1975)
Hoet, P., Fraselle, G., Cocito, C.: Recombination-type transfer of viral DNA during bacteriophage 2C replication in B. subtilis. J. Virol. 17, 718–726 (1976)
Kainuma-Kuroda, R., Okazaki, R.: Mechanism of DNA chain growth. XII. Asymmetry of replication of P2 phage DNA. J. Mol. Biol. 94, 213–228 (1975)
Kurosawa, Y., Okazaki, R.: Mechanism of DNA chain growth. XIII. Evidence for discontinuous replication of both strands of P2 phage DNA. J. Mol. Biol. 94, 229–241 (1975)
Louarn, J.M., Bird, R.E.: Size distribution and molecular polarity of newly replicated DNA in E. coli. Proc. Natl. Acad. Sci. U.S.A. 71, 329–333 (1974)
May, P., May, E., Granboulan, P., Granboulan, N., Marmur, J.: Ultrastructure du bactériophage 2C et propriétés de son DNA. Ann. Inst. Pasteur 115, 1029–1046 (1968)
Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K., Kainuma, R., Sugino, A., Iwatsuki, N.: In vivo mechanism of DNA chain growth. Cold Spring Harbor Symp. Quant. Biol. 33, 129–143 (1968)
Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K., Sugino, A.: Mechanism of DNA chain growth. I. Possible discontinuity and unusual secondary structure of newly synthesized chains. Proc. Natl. Acad. Sci. U.S.A. 59, 598–605 (1968)
Okazaki, R., Sugimoto, K., Okazaki, T., Imae, Y., Sugino, A.: DNA chain growth; in vivo and in vitro synthesis in a DNA polymerase — negative mutant of E. coli. Nature 228, 223–226 (1970)
Polsinelli, M., Milanesi, G., Ganesan, A.T.: Short fragments from both complementary strands in the newly replicated DNA of bacteriophage SPPI. Science 166, 243–245 (1969)
Schnös, M., Inman, R.B.: Position of branch points in replicating λ DNA. J. Mol. Biol. 51, 61–73 (1970)
Sternglanz, R., Wang, H.F., Donegan, J.J.: Evidence that both growing DNA chains at a replication fork are synthesized discontinuously. Biochemistry 15, 1838–1843 (1976)
Stevens, W.F., Adhya, S., Szybalski, W.: Origin and bidirectional orientation of DNA replication in coliphage lambda. In: The bacteriophage lambda (Hershey, A.D. ed.), pp 515–533. New York: Cold Spring Harbor Laboratory 1971
Studier, F.W.: Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11, 373–390 (1965)
Sugimoto, K., Okazaki, T., Imae, Y., Okazaki, R.: Mechanism of DNA chain growth. III. Equal annealing of T4 nascent short DNA chains with the separated complementary strands of the phage DNA. Proc. Natl. Acad. Sci. U.S.A. 63, 1343–1350 (1969)
Truffaut, N.: Isolement et propriétés des chaînes du DNA du bactériophage 2C. Eur. J. Biochem. 13, 438–446 (1970)
Tye, B.K., Lehman, I.R.: Excision repair of uracil incorporated in DNA as a result of a defect in dUTPase. J. Mol. Biol. 117, 293–306 (1977)
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Communicated by H.G. Wittmann
This article is dedicated to the memory of Georges Fraselle (1949–1977).
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Hoet, P.P., Fraselle, G. & Cocito, C. Discontinuous duplication of both strands of virus 2C DNA. Molec. Gen. Genet. 171, 43–51 (1979). https://doi.org/10.1007/BF00274013
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DOI: https://doi.org/10.1007/BF00274013