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Nucleic Acids Research, 1990, Vol. 18, No. 18 5449-5455
© 1990

MOLECULAR BIOLOGY

Assembly of correctly spaced chromatin in a nuclear extract from Xenopus laevis oocytes

Giovanna Sessa and Ida Ruberti*

Centro di studio per gli Acidi Nucleici del CNR, c/o Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza P le Aldo Moro 5, 00185 Rome, Italy

*To whom correspondence should be addressed

Received June 13, 1990. Revised August 20, 1990. Accepted August 20, 1990.

Assembly of nucleosomes on relaxed, covalently closed DNA has been studied in a nuclear extract of Xenopus laevis oocytes. Nucleosomes containing the four histones H3, H4, H2A and H2B but lacking histone H1 are readily assembled on the DNA. The pattern of micrococcal nuclease digestion shows that the nucleosomes assembled in the absence of ATP and Mg (II) are closely packed, with a periodicity of 150 base pairs (bp). In contrast, in the presence of ATP and Mg (II) the spacing of the nucleosomes is 180 bp, similar to that observed for nucleosomes assembled on DNA microinjected into oocyte nuclei. The ATP and Mg (II) requirements for the assembly of correctly spaced nucleosomes are unrelated to the activity of the ATP and Mg (II) dependent DNA topoisomerase II in the extract; addition of specific inhibitors of eukaryotic DNA topoisomerase II has no effect on the spacing of the reconstituted nucleosomes. The ATP requirement in the assembly of correctly spaced nucleosomes can be substituted by adenosine 5'-O-3'-thiotriphosphate ({gamma}-S-ATP) but not by adenyl-5'-yl imidodiphosphate (AMP-P-(NH)-P).


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