Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1992 Feb 25;20(4):889–895. doi: 10.1093/nar/20.4.889

In vitro chromatin assembly promoted by the Xenopus laevis S-150 cell-free extract is enhanced by treatment with RNase A.

J M Sekiguchi 1, E B Kmiec 1
PMCID: PMC312033  PMID: 1371870

Abstract

Cell-free extracts employed as chromatin assembly systems contain a myriad of proteins, polyanions and nucleic acids. The roles of ATP, MgCl2 and other cofactors in the catalysis of nucleosome formation by the Xenopus laevis oocyte S-150 have yet to be established unequivocally. In this study we examine the influence of RNA in the assembly process. Under reaction conditions that inhibit nucleosome formation (+ EDTA), pretreatment of the extract with RNase A revives the chromatin assembly machinery while the rate of DNA supercoiling is stimulated significantly. Addition of purified RNA blocks DNA supercoiling. Taken together, these data suggest that the parameters surrounding in vitro chromatin assembly are variable and subject to modulation by endogenous factors.

Full text

PDF
889

Images in this article

890Image
on p.890
891Image
on p.891
892Image
on p.892
893Image
on p.893
893Image
on p.893
894Image
on p.894

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. Banerjee S., Cantor C. R. Nucleosome assembly of simian virus 40 DNA in a mammalian cell extract. Mol Cell Biol. 1990 Jun;10(6):2863–2873. doi: 10.1128/mcb.10.6.2863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Close T. J., Christmann J. L., Rodriguez R. L. M13 bacteriophage and pUC plasmids containing DNA inserts but still capable of beta-galactosidase alpha-complementation. Gene. 1983 Aug;23(2):131–136. doi: 10.1016/0378-1119(83)90044-6. [DOI] [PubMed] [Google Scholar]
  5. Germond J. E., Rouvière-Yaniv J., Yaniv M., Brutlag D. Nicking-closing enzyme assembles nucleosome-like structures in vitro. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3779–3783. doi: 10.1073/pnas.76.8.3779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Kmiec E. B., Sekiguchi J. M., Cole A. D. Studies on the ATP requirements of in vitro chromatin assembly. Biochem Cell Biol. 1989 Aug;67(8):443–454. doi: 10.1139/o89-070. [DOI] [PubMed] [Google Scholar]
  8. Laskey R. A., Earnshaw W. C. Nucleosome assembly. Nature. 1980 Aug 21;286(5775):763–767. doi: 10.1038/286763a0. [DOI] [PubMed] [Google Scholar]
  9. Laskey R. A., Honda B. M., Mills A. D., Finch J. T. Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA. Nature. 1978 Oct 5;275(5679):416–420. doi: 10.1038/275416a0. [DOI] [PubMed] [Google Scholar]
  10. Manning G. S. The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides. Q Rev Biophys. 1978 May;11(2):179–246. doi: 10.1017/s0033583500002031. [DOI] [PubMed] [Google Scholar]
  11. Nelson T., Wiegand R., Brutlag D. Ribonucleic acid and other polyanions facilitate chromatin assembly in vitro. Biochemistry. 1981 Apr 28;20(9):2594–2601. doi: 10.1021/bi00512a035. [DOI] [PubMed] [Google Scholar]
  12. Newton I., Rinke J., Brimacombe R. Random exchange of ribosomal proteins in EDTA sub-particles. FEBS Lett. 1975 Mar 1;51(1):215–218. doi: 10.1016/0014-5793(75)80890-8. [DOI] [PubMed] [Google Scholar]
  13. Ruberti I., Worcel A. Mechanism of chromatin assembly in Xenopus oocytes. J Mol Biol. 1986 Jun 5;189(3):457–476. doi: 10.1016/0022-2836(86)90317-7. [DOI] [PubMed] [Google Scholar]
  14. Sekiguchi J. A., Kmiec E. B. Reaction parameters of TFIIIA-induced supercoiling catalyzed by a Xenopus laevis cell-free extract. Nucleic Acids Res. 1990 Feb 25;18(4):1021–1029. doi: 10.1093/nar/18.4.1021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sekiguchi J. M., Kmiec E. B. An analysis of transcription factor TFIIIA-mediated DNA supercoiling. DNA Cell Biol. 1991 Apr;10(3):223–232. doi: 10.1089/dna.1991.10.223. [DOI] [PubMed] [Google Scholar]
  16. Sekiguchi J. M., Swank R. A., Kmiec E. B. Changes in DNA topology can modulate in vitro transcription of certain RNA polymerase III genes. Mol Cell Biochem. 1989 Feb 21;85(2):123–133. doi: 10.1007/BF00577108. [DOI] [PubMed] [Google Scholar]
  17. Shimamura A., Worcel A. The assembly of regularly spaced nucleosomes in the Xenopus oocyte S-150 extract is accompanied by deacetylation of histone H4. J Biol Chem. 1989 Aug 25;264(24):14524–14530. [PubMed] [Google Scholar]
  18. 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]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES