Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1990 Sep;9(9):2911–2922. doi: 10.1002/j.1460-2075.1990.tb07482.x

Nucleosome assembly in mammalian cell extracts before and after DNA replication.

C Gruss 1, C Gutierrez 1, W C Burhans 1, M L DePamphilis 1, T Koller 1, J M Sogo 1
PMCID: PMC552007  PMID: 2167837

Abstract

Protein-free DNA in a cytosolic extract supplemented with SV40 large T-antigen (T-Ag), is assembled into chromatin structure when nuclear extract is added. This assembly was monitored by topoisomer formation, micrococcal nuclease digestion and psoralen crosslinking of the DNA. Plasmids containing SV40 sequences (ori- and ori+) were assembled into chromatin with similar efficiencies whether T-Ag was present or not. Approximately 50-80% of the number of nucleosomes in vivo could be assembled in vitro; however, the kinetics of assembly differed on replicated and unreplicated molecules. In replicative intermediates, nucleosomes were observed on both the pre-replicated and post-replicated portions. We conclude that the extent of nucleosome assembly in mammalian cell extracts is not dependent upon DNA replication, in contrast to previous suggestions. However, the highly sensitive psoralen assay revealed that DNA replication appears to facilitate precise folding of DNA in the nucleosome.

Full text

PDF
2911

Images in this article

2912Fig. 1.
on p.2912
2912Fig. 2.
on p.2912
2913Fig. 3.
on p.2913
2914Fig. 4.
on p.2914
2914Fig. 5.
on p.2914
2915Fig. 6.
on p.2915
2916Fig. 7.
on p.2916
2917Fig. 8.
on p.2917
2918Fig. 9.
on p.2918

Selected References

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

  1. Almouzni G., Méchali M. Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity. EMBO J. 1988 Dec 20;7(13):4355–4365. doi: 10.1002/j.1460-2075.1988.tb03334.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Almouzni G., Méchali M. Assembly of spaced chromatin promoted by DNA synthesis in extracts from Xenopus eggs. EMBO J. 1988 Mar;7(3):665–672. doi: 10.1002/j.1460-2075.1988.tb02861.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Avemann K., Knippers R., Koller T., Sogo J. M. Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks. Mol Cell Biol. 1988 Aug;8(8):3026–3034. doi: 10.1128/mcb.8.8.3026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barsoum J., Berg P. Simian virus 40 minichromosomes contain torsionally strained DNA molecules. Mol Cell Biol. 1985 Nov;5(11):3048–3057. doi: 10.1128/mcb.5.11.3048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birnboim H. C. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983;100:243–255. doi: 10.1016/0076-6879(83)00059-2. [DOI] [PubMed] [Google Scholar]
  6. Bonne-Andrea C., Wong M. L., Alberts B. M. In vitro replication through nucleosomes without histone displacement. Nature. 1990 Feb 22;343(6260):719–726. doi: 10.1038/343719a0. [DOI] [PubMed] [Google Scholar]
  7. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  8. Cech T. R., Potter D., Pardue M. L. Chromatin structure in living cells. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 1):191–198. doi: 10.1101/sqb.1978.042.01.021. [DOI] [PubMed] [Google Scholar]
  9. Cereghini S., Yaniv M. Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication. EMBO J. 1984 Jun;3(6):1243–1253. doi: 10.1002/j.1460-2075.1984.tb01959.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Challberg M. D., Kelly T. J. Animal virus DNA replication. Annu Rev Biochem. 1989;58:671–717. doi: 10.1146/annurev.bi.58.070189.003323. [DOI] [PubMed] [Google Scholar]
  11. Cheng L., Kelly T. J. Transcriptional activator nuclear factor I stimulates the replication of SV40 minichromosomes in vivo and in vitro. Cell. 1989 Nov 3;59(3):541–551. doi: 10.1016/0092-8674(89)90037-8. [DOI] [PubMed] [Google Scholar]
  12. Conconi A., Losa R., Koller T., Sogo J. M. Psoralen-crosslinking of soluble and of H1-depleted soluble rat liver chromatin. J Mol Biol. 1984 Oct 5;178(4):920–928. doi: 10.1016/0022-2836(84)90319-x. [DOI] [PubMed] [Google Scholar]
  13. Conconi A., Widmer R. M., Koller T., Sogo J. M. Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle. Cell. 1989 Jun 2;57(5):753–761. doi: 10.1016/0092-8674(89)90790-3. [DOI] [PubMed] [Google Scholar]
  14. Cusick M. E., DePamphilis M. L., Wassarman P. M. Dispersive segregation of nucleosomes during replication of simian virus 40 chromosomes. J Mol Biol. 1984 Sep 15;178(2):249–271. doi: 10.1016/0022-2836(84)90143-8. [DOI] [PubMed] [Google Scholar]
  15. Cusick M. E., Wassarman P. M., DePamphilis M. L. Application of nucleases to visualizing chromatin organization at replication forks. Methods Enzymol. 1989;170:290–316. doi: 10.1016/0076-6879(89)70053-7. [DOI] [PubMed] [Google Scholar]
  16. De Bernardin W., Koller T., Sogo J. M. Structure of in-vivo transcribing chromatin as studied in simian virus 40 minichromosomes. J Mol Biol. 1986 Oct 5;191(3):469–482. doi: 10.1016/0022-2836(86)90142-7. [DOI] [PubMed] [Google Scholar]
  17. Decker R. S., Yamaguchi M., Possenti R., Bradley M. K., DePamphilis M. L. In vitro initiation of DNA replication in simian virus 40 chromosomes. J Biol Chem. 1987 Aug 5;262(22):10863–10872. [PubMed] [Google Scholar]
  18. Dilworth S. M., Black S. J., Laskey R. A. Two complexes that contain histones are required for nucleosome assembly in vitro: role of nucleoplasmin and N1 in Xenopus egg extracts. Cell. 1987 Dec 24;51(6):1009–1018. doi: 10.1016/0092-8674(87)90587-3. [DOI] [PubMed] [Google Scholar]
  19. Dodson M., Dean F. B., Bullock P., Echols H., Hurwitz J. Unwinding of duplex DNA from the SV40 origin of replication by T antigen. Science. 1987 Nov 13;238(4829):964–967. doi: 10.1126/science.2823389. [DOI] [PubMed] [Google Scholar]
  20. Fotedar R., Roberts J. M. Multistep pathway for replication-dependent nucleosome assembly. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6459–6463. doi: 10.1073/pnas.86.17.6459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Germond J. E., Hirt B., Oudet P., Gross-Bellark M., Chambon P. Folding of the DNA double helix in chromatin-like structures from simian virus 40. Proc Natl Acad Sci U S A. 1975 May;72(5):1843–1847. doi: 10.1073/pnas.72.5.1843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Glikin G. C., Ruberti I., Worcel A. Chromatin assembly in Xenopus oocytes: in vitro studies. Cell. 1984 May;37(1):33–41. doi: 10.1016/0092-8674(84)90298-8. [DOI] [PubMed] [Google Scholar]
  23. Griffith J. D. Chromatin structure: deduced from a minichromosome. Science. 1975 Mar 28;187(4182):1202–1203. doi: 10.1126/science.187.4182.1202. [DOI] [PubMed] [Google Scholar]
  24. Gruss C., Baumann E., Knippers R. DNA binding properties of a mutant T antigen from the simian virus 40-transformed human cell line simian virus 80. J Virol. 1984 Jun;50(3):943–946. doi: 10.1128/jvi.50.3.943-946.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Guo Z. S., Gutierrez C., Heine U., Sogo J. M., Depamphilis M. L. Origin auxiliary sequences can facilitate initiation of simian virus 40 DNA replication in vitro as they do in vivo. Mol Cell Biol. 1989 Sep;9(9):3593–3602. doi: 10.1128/mcb.9.9.3593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gutierrez C., Guo Z. S., Roberts J., DePamphilis M. L. Simian virus 40 origin auxiliary sequences weakly facilitate T-antigen binding but strongly facilitate DNA unwinding. Mol Cell Biol. 1990 Apr;10(4):1719–1728. doi: 10.1128/mcb.10.4.1719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Handeli S., Klar A., Meuth M., Cedar H. Mapping replication units in animal cells. Cell. 1989 Jun 16;57(6):909–920. doi: 10.1016/0092-8674(89)90329-2. [DOI] [PubMed] [Google Scholar]
  28. Hanson C. V., Shen C. K., Hearst J. E. Cross-linking of DNA in situ as a probe for chromatin structure. Science. 1976 Jul 2;193(4247):62–64. doi: 10.1126/science.935855. [DOI] [PubMed] [Google Scholar]
  29. Igo-Kemenes T., Hörz W., Zachau H. G. Chromatin. Annu Rev Biochem. 1982;51:89–121. doi: 10.1146/annurev.bi.51.070182.000513. [DOI] [PubMed] [Google Scholar]
  30. Jackson V. Deposition of newly synthesized histones: new histones H2A and H2B do not deposit in the same nucleosome with new histones H3 and H4. Biochemistry. 1987 Apr 21;26(8):2315–2325. doi: 10.1021/bi00382a037. [DOI] [PubMed] [Google Scholar]
  31. Jackson V. In vivo studies on the dynamics of histone-DNA interaction: evidence for nucleosome dissolution during replication and transcription and a low level of dissolution independent of both. Biochemistry. 1990 Jan 23;29(3):719–731. doi: 10.1021/bi00455a019. [DOI] [PubMed] [Google Scholar]
  32. Jakobovits E. B., Bratosin S., Aloni Y. A nucleosome-free region in SV40 minichromosomes. Nature. 1980 May 22;285(5762):263–265. doi: 10.1038/285263a0. [DOI] [PubMed] [Google Scholar]
  33. Kelly T. J. SV40 DNA replication. J Biol Chem. 1988 Dec 5;263(34):17889–17892. [PubMed] [Google Scholar]
  34. Koller T., Kübler O., Portmann R., Sogo J. M. High resolution physical mapping of specific binding sites of Escherichia coli RNA polymerase on the DNA of bacteriophage T7 . J Mol Biol. 1978 Mar 25;120(1):121–131. doi: 10.1016/0022-2836(78)90298-x. [DOI] [PubMed] [Google Scholar]
  35. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  36. Laskey R. A., Mills A. D., Morris N. R. Assembly of SV40 chromatin in a cell-free system from Xenopus eggs. Cell. 1977 Feb;10(2):237–243. doi: 10.1016/0092-8674(77)90217-3. [DOI] [PubMed] [Google Scholar]
  37. Li J. J., Kelly T. J. Simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6973–6977. doi: 10.1073/pnas.81.22.6973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Li J. J., Kelly T. J. Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication. Mol Cell Biol. 1985 Jun;5(6):1238–1246. doi: 10.1128/mcb.5.6.1238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Lucchini R., Pauli U., Braun R., Koller T., Sogo J. M. Structure of the extrachromosomal ribosomal RNA chromatin of Physarum polycephalum. J Mol Biol. 1987 Aug 20;196(4):829–843. doi: 10.1016/0022-2836(87)90408-6. [DOI] [PubMed] [Google Scholar]
  40. Lusky M., Botchan M. Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences. Nature. 1981 Sep 3;293(5827):79–81. doi: 10.1038/293079a0. [DOI] [PubMed] [Google Scholar]
  41. Martínez-Salas E., Linney E., Hassell J., DePamphilis M. L. The need for enhancers in gene expression first appears during mouse development with formation of the zygotic nucleus. Genes Dev. 1989 Oct;3(10):1493–1506. doi: 10.1101/gad.3.10.1493. [DOI] [PubMed] [Google Scholar]
  42. Nelson T., Hsieh T. S., Brutlag D. Extracts of Drosophila embryos mediate chromatin assembly in vitro. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5510–5514. doi: 10.1073/pnas.76.11.5510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Ohlenbusch H. H., Olivera B. M., Tuan D., Davidson N. Selective dissociation of histones from calf thymus nucleoprotein. J Mol Biol. 1967 Apr 28;25(2):299–315. doi: 10.1016/0022-2836(67)90143-x. [DOI] [PubMed] [Google Scholar]
  44. Oudet P., Weiss E., Regnier E. Preparation of simian virus 40 minichromosomes. Methods Enzymol. 1989;170:14–25. doi: 10.1016/0076-6879(89)70040-9. [DOI] [PubMed] [Google Scholar]
  45. Peden K. W., Pipas J. M., Pearson-White S., Nathans D. Isolation of mutants of an animal virus in bacteria. Science. 1980 Sep 19;209(4463):1392–1396. doi: 10.1126/science.6251547. [DOI] [PubMed] [Google Scholar]
  46. Rhodes D., Laskey R. A. Assembly of nucleosomes and chromatin in vitro. Methods Enzymol. 1989;170:575–585. doi: 10.1016/0076-6879(89)70065-3. [DOI] [PubMed] [Google Scholar]
  47. Robinson G. W., Hallick L. M. Mapping the in vivo arrangement of nucleosomes on simian virus 40 chromatin by the photoaddition of radioactive hydroxymethyltrimethylpsoralen. J Virol. 1982 Jan;41(1):78–87. doi: 10.1128/jvi.41.1.78-87.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Ryoji M., Worcel A. Structure of the two distinct types of minichromosomes that are assembled on DNA injected in Xenopus oocytes. Cell. 1985 Apr;40(4):923–932. doi: 10.1016/0092-8674(85)90352-6. [DOI] [PubMed] [Google Scholar]
  49. Saragosti S., Moyne G., Yaniv M. Absence of nucleosomes in a fraction of SV40 chromatin between the origin of replication and the region coding for the late leader RNA. Cell. 1980 May;20(1):65–73. doi: 10.1016/0092-8674(80)90235-4. [DOI] [PubMed] [Google Scholar]
  50. Scott W. A., Wigmore D. J. Sites in simian virus 40 chromatin which are preferentially cleaved by endonucleases. Cell. 1978 Dec;15(4):1511–1518. doi: 10.1016/0092-8674(78)90073-9. [DOI] [PubMed] [Google Scholar]
  51. Seidman M. M., Levine A. J., Weintraub H. The asymmetric segregation of parental nucleosomes during chrosome replication. Cell. 1979 Oct;18(2):439–449. doi: 10.1016/0092-8674(79)90063-1. [DOI] [PubMed] [Google Scholar]
  52. Shimamura A., Jessee B., Worcel A. Assembly of chromatin with oocyte extracts. Methods Enzymol. 1989;170:603–612. doi: 10.1016/0076-6879(89)70067-7. [DOI] [PubMed] [Google Scholar]
  53. Simanis V., Lane D. P. An immunoaffinity purification procedure for SV40 large T antigen. Virology. 1985 Jul 15;144(1):88–100. doi: 10.1016/0042-6822(85)90308-3. [DOI] [PubMed] [Google Scholar]
  54. Smith S., Stillman B. Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro. Cell. 1989 Jul 14;58(1):15–25. doi: 10.1016/0092-8674(89)90398-x. [DOI] [PubMed] [Google Scholar]
  55. Sogo J. M., Ness P. J., Widmer R. M., Parish R. W., Koller T. Psoralen-crosslinking of DNA as a probe for the structure of active nucleolar chromatin. J Mol Biol. 1984 Oct 5;178(4):897–919. doi: 10.1016/0022-2836(84)90318-8. [DOI] [PubMed] [Google Scholar]
  56. Sogo J. M., Stahl H., Koller T., Knippers R. Structure of replicating simian virus 40 minichromosomes. The replication fork, core histone segregation and terminal structures. J Mol Biol. 1986 May 5;189(1):189–204. doi: 10.1016/0022-2836(86)90390-6. [DOI] [PubMed] [Google Scholar]
  57. Stahl H., Dröge P., Knippers R. DNA helicase activity of SV40 large tumor antigen. EMBO J. 1986 Aug;5(8):1939–1944. doi: 10.1002/j.1460-2075.1986.tb04447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Stillman B. W., Gluzman Y. Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. Mol Cell Biol. 1985 Aug;5(8):2051–2060. doi: 10.1128/mcb.5.8.2051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Stillman B. Chromatin assembly during SV40 DNA replication in vitro. Cell. 1986 May 23;45(4):555–565. doi: 10.1016/0092-8674(86)90287-4. [DOI] [PubMed] [Google Scholar]
  60. Stillman B. Initiation of eukaryotic DNA replication in vitro. Annu Rev Cell Biol. 1989;5:197–245. doi: 10.1146/annurev.cb.05.110189.001213. [DOI] [PubMed] [Google Scholar]
  61. Stockley P. G., Thomas J. O. A nucleosome-like particle containing an octamer of the arginine-rich histones H3 and H4. FEBS Lett. 1979 Mar 1;99(1):129–135. doi: 10.1016/0014-5793(79)80264-1. [DOI] [PubMed] [Google Scholar]
  62. Sundin O., Varshavsky A. Staphylococcal nuclease makes a single non-random cut in the simian virus 40 viral minichromosome. J Mol Biol. 1979 Aug 15;132(3):535–546. doi: 10.1016/0022-2836(79)90274-2. [DOI] [PubMed] [Google Scholar]
  63. Thoma F., Losa R., Koller T. Involvement of the domains of histones H1 and H5 in the structural organization of soluble chromatin. J Mol Biol. 1983 Jul 5;167(3):619–640. doi: 10.1016/s0022-2836(83)80102-8. [DOI] [PubMed] [Google Scholar]
  64. Varshavsky A. J., Sundin O. H., Bohn M. J. SV40 viral minichromosome: preferential exposure of the origin of replication as probed by restriction endonucleases. Nucleic Acids Res. 1978 Oct;5(10):3469–3477. doi: 10.1093/nar/5.10.3469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Varshavsky A. J., Sundin O., Bohn M. A stretch of "late" SV40 viral DNA about 400 bp long which includes the origin of replication is specifically exposed in SV40 minichromosomes. Cell. 1979 Feb;16(2):453–466. doi: 10.1016/0092-8674(79)90021-7. [DOI] [PubMed] [Google Scholar]
  66. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  67. Wobbe C. R., Dean F., Weissbach L., Hurwitz J. In vitro replication of duplex circular DNA containing the simian virus 40 DNA origin site. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5710–5714. doi: 10.1073/pnas.82.17.5710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Yamaguchi M., DePamphilis M. L. DNA binding site for a factor(s) required to initiate simian virus 40 DNA replication. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1646–1650. doi: 10.1073/pnas.83.6.1646. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES