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. 1972 Aug;69(8):2300–2305. doi: 10.1073/pnas.69.8.2300

Sites of Replication of Chromosomal DNA in a Eukaryotic Cell

Stanislav Fakan 1, Gail N Turner 1, Joseph S Pagano 1,*, Ronald Hancock 1
PMCID: PMC426922  PMID: 4506099

Abstract

In mouse cells (line P815), newly synthesized DNA labeled for 20-30 sec during exponential growth is found by electron microscope autoradiography at sites throughout the cell nucleus. These sites are relatively more concentrated in the peripheral region of the nucleus (averaged over a random population of S-phase cells), probably reflecting a higher local concentration of DNA in this region. Newly synthesized DNA is not preferentially associated with purified nuclear envelopes, but is found in a fraction of the chromosomal deoxynucleoprotein whose buoyant density in CsCl after formaldehyde treatment is about 1% lower than that of the deoxynucleoprotein peak. Kinetics experiments suggest that this material is a precursor of mature deoxynucleoprotein; it may represent regions of deoxynucleoprotein containing replicating DNA and the additional proteins involved in DNA replication. Other complexes of newly replicated DNA that are found in the interphase after phenol extraction of nuclei are formed during the extraction procedure, probably due to the partially single-stranded nature of replicating DNA, and do not appear to exist in vivo.

Keywords: chromatin, nuclear membrane, replication complex, autoradiography

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BACHMANN L., SALPETER M. M. AUTORADIOGRAPHY WITH THE ELECTRON MICROSCOPE; A QUANTITATIVE EVALUATION. Lab Invest. 1965 Jun;14:1041–1053. [PubMed] [Google Scholar]
  2. BEN-PORAT T., STERE A., KAPLAN A. S. The separation of nascent deoxyribonucleic acid from the remainder of the cellular deoxyribonucleic acid. Biochim Biophys Acta. 1962 Jul 9;61:150–152. doi: 10.1016/0926-6550(62)90045-2. [DOI] [PubMed] [Google Scholar]
  3. Berger H., Jr, Irvin J. L. Changes in the physical state of DNA during replication in regenerating liver of the rat. Proc Natl Acad Sci U S A. 1970 Jan;65(1):152–159. doi: 10.1073/pnas.65.1.152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cairns J. Autoradiography of HeLa cell DNA. J Mol Biol. 1966 Jan;15(1):372–373. doi: 10.1016/s0022-2836(66)80233-4. [DOI] [PubMed] [Google Scholar]
  5. Comings D. E., Kakefuda T. Initiation of deoxyribonucleic acid replication at the nuclear membrane in human cells. J Mol Biol. 1968 Apr 14;33(1):225–229. doi: 10.1016/0022-2836(68)90290-8. [DOI] [PubMed] [Google Scholar]
  6. Delius H., Howe C., Kozinski A. W. Structure of the replicating DNA from bacteriophage T4. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3049–3053. doi: 10.1073/pnas.68.12.3049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Deumling B., Franke W. W. Nuclear membranes from mammalian liver, V. On the question of DNA polymerase activities associated with the nuclear envelope. Hoppe Seylers Z Physiol Chem. 1972 Mar;353(3):287–297. doi: 10.1515/bchm2.1972.353.1.287. [DOI] [PubMed] [Google Scholar]
  8. Franke W. W., Deumling B., Baerbelermen, Jarasch E. D., Kleinig H. Nuclear membranes from mammalian liver. I. Isolation procedure and general characterization. J Cell Biol. 1970 Aug;46(2):379–395. doi: 10.1083/jcb.46.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Friedman D. L., Mueller G. C. Studies on the nature of replicating DNA of HeLa cells. Biochim Biophys Acta. 1969 Jan 21;174(1):253–263. doi: 10.1016/0005-2787(69)90249-4. [DOI] [PubMed] [Google Scholar]
  10. Habener J. F., Bynum B. S., Shack J. Changes in the sedimentation properties of HeLa cell DNA and nucleoprotein during replication. Biochim Biophys Acta. 1969 Dec 16;195(2):484–493. doi: 10.1016/0005-2787(69)90655-8. [DOI] [PubMed] [Google Scholar]
  11. Habener J. F., Bynum B. S., Shack J. Destabilized secondary structure of newly replicated HeLa DNA. J Mol Biol. 1970 Apr 14;49(1):157–170. doi: 10.1016/0022-2836(70)90383-9. [DOI] [PubMed] [Google Scholar]
  12. Hanaoka F., Yamada M. Localization of the replication point of mammalian cell DNA at the membrane. Biochem Biophys Res Commun. 1971 Feb 19;42(4):647–653. doi: 10.1016/0006-291x(71)90537-7. [DOI] [PubMed] [Google Scholar]
  13. Hancock R. Conservation of histones in chromatin during growth and mitosis in vitro. J Mol Biol. 1969 Mar 28;40(3):457–466. doi: 10.1016/0022-2836(69)90165-x. [DOI] [PubMed] [Google Scholar]
  14. Kashnig D. M., Kasper C. B. Isolation, morphology, and composition of the nuclear membrane from rat liver. J Biol Chem. 1969 Jul 25;244(14):3786–3792. [PubMed] [Google Scholar]
  15. Kay R. R., Haines M. E., Johnston I. R. Late replication of the DNA associated with the nuclear membrane. FEBS Lett. 1971 Sep 1;16(4):233–236. doi: 10.1016/0014-5793(71)80358-7. [DOI] [PubMed] [Google Scholar]
  16. Knippers R., Sinsheimer R. L. Process of infection with bacteriophage phiX174. XX. Attachment of the parental DNA of bacteriophage phiX174 to a fast-sedimenting cell component. J Mol Biol. 1968 May 28;34(1):17–29. doi: 10.1016/0022-2836(68)90231-3. [DOI] [PubMed] [Google Scholar]
  17. Kolber A. R., Sly W. S. Association of lambda bacteriophage DNA with a rapidly sedimenting Escherichia coli component. Virology. 1971 Dec;46(3):638–654. doi: 10.1016/0042-6822(71)90067-5. [DOI] [PubMed] [Google Scholar]
  18. Mizuno N. S., Stoops C. E., Peiffer R. L., Jr Nature of the DNA associated with the nuclear envelope of regenerating liver. J Mol Biol. 1971 Aug 14;59(3):517–525. doi: 10.1016/0022-2836(71)90315-9. [DOI] [PubMed] [Google Scholar]
  19. Monneron A., Moulé Y. Critical evaluation of specificity in electron microscopical radioautography in animal tissues. Exp Cell Res. 1969 Aug;56(2):179–193. doi: 10.1016/0014-4827(69)90001-9. [DOI] [PubMed] [Google Scholar]
  20. Nancock R. Separation by equilibrium centrifugation in CsC1 gradients of density--labelled and normal deoxyribonucleoprotein from chromatin. J Mol Biol. 1970 Mar 14;48(2):357–360. doi: 10.1016/0022-2836(70)90167-1. [DOI] [PubMed] [Google Scholar]
  21. O'Brien R. L., Sanyal A. B., Stanton R. H. Association of DNA replication with the nuclear membrane of HeLa cells. Exp Cell Res. 1972 Jan;70(1):106–112. doi: 10.1016/0014-4827(72)90187-5. [DOI] [PubMed] [Google Scholar]
  22. Ockey C. H. Distribution of DNA replicator sites in mammalian nuclei. II. Effects of prolonged inhibition of DNA synthesis. Exp Cell Res. 1972 Jan;70(1):203–213. doi: 10.1016/0014-4827(72)90198-x. [DOI] [PubMed] [Google Scholar]
  23. Painter R. B., Schaefer A. State of newly synthesized HeLa DNA. Nature. 1969 Mar 29;221(5187):1215–1217. doi: 10.1038/2211215a0. [DOI] [PubMed] [Google Scholar]
  24. Paoletti C., Dutheillet-Lamonthézie N., Jeanteur P., Obrenovitch A. Mise en évidence et étude cinétique des formes replicatives du DNA dans les cellules animales. Biochim Biophys Acta. 1967 Dec 19;149(2):435–450. [PubMed] [Google Scholar]
  25. Pearson G. D., Hanawalt P. C. Isolation of DNA replication complexes from uninfected and adenovirus-infected HeLa cells. J Mol Biol. 1971 Nov 28;62(1):65–80. doi: 10.1016/0022-2836(71)90131-8. [DOI] [PubMed] [Google Scholar]
  26. Probst H. Untersuchungen zur Charakterisierung der durch NaCl eluierbaren Methylalbumin-Kieselgur-Fraktionen neusynthetisierter DNA aus Säugetierzellen. Hoppe Seylers Z Physiol Chem. 1971 May;352(5):748–756. [PubMed] [Google Scholar]
  27. Rasmussen R. E., Painter R. B. Radiation-stimulated DNA synthesis in cultured mammalian cells. J Cell Biol. 1966 Apr;29(1):11–19. doi: 10.1083/jcb.29.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schindler R., Ramseier L., Schaer J. C., Grieder A. Studies on the division cycle of mammalian cells. 3. Preparation of synchronously dividing cell populations by isotonic sucrose gradient centrifugation. Exp Cell Res. 1970 Jan;59(1):90–96. doi: 10.1016/0014-4827(70)90627-0. [DOI] [PubMed] [Google Scholar]
  29. Sueoka N., Quinn W. G. Membrane attachment of the chromosome replication origin in Bacillus subtilis. Cold Spring Harb Symp Quant Biol. 1968;33:695–705. doi: 10.1101/sqb.1968.033.01.078. [DOI] [PubMed] [Google Scholar]
  30. Taylor J. H. Rates of chain growth and units of replication in DNA of mammalian chromosomes. J Mol Biol. 1968 Feb 14;31(3):579–594. doi: 10.1016/0022-2836(68)90429-4. [DOI] [PubMed] [Google Scholar]
  31. Williams C. A., Ockey C. H. Distribution of DNA replicator sites in mammalian nuclei after different methods of cell synchronization. Exp Cell Res. 1970 Dec;63(2):365–372. doi: 10.1016/0014-4827(70)90224-7. [DOI] [PubMed] [Google Scholar]
  32. Yasmineh W. G., Yunis J. J. Localization of mouse satellite DNA in constitutive heterochromatin. Exp Cell Res. 1970 Jan;59(1):69–75. doi: 10.1016/0014-4827(70)90624-5. [DOI] [PubMed] [Google Scholar]

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