Functional analysis of nucleosome assembly protein, NAP-1. The negatively charged COOH-terminal region is not necessary for the intrinsic assembly activity

J Biol Chem. 1992 Oct 15;267(29):20980-6.

Abstract

A nucleosome assembly protein (NAP-1) of Saccharomyces cerevisiae facilitates the association of histones with DNA to form nucleosomes in vitro at physiological ionic conditions. The cloned gene was expressed in Escherichia coli using a T7 expression system, and the protein (417 amino acid residues) was purified by Mono Q column chromatography. Various deletion fragments of NAP-1 protein were also produced, and their nucleosome assembly activity was examined by supercoiling assay. The internal fragment containing the residues 43-365 was necessary and sufficient for the activity, and a long stretch of negatively charged region near the carboxyl terminus was dispensable. This minimal size fragment could form the 12 S NAP-1-histone complex as the whole protein could, whereas deleted fragments on either side could bind with core histones only to form aggregates.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Bacteriophage T7 / genetics
  • Base Sequence
  • Cell Cycle Proteins
  • Chromatography, Ion Exchange
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Fungal Proteins / genetics*
  • Fungal Proteins / isolation & purification
  • Fungal Proteins / metabolism
  • Genes, Fungal*
  • Histones / metabolism
  • Immunoblotting
  • Mice
  • Molecular Sequence Data
  • Nuclear Proteins
  • Nucleosome Assembly Protein 1
  • Plasmids
  • Proteins / genetics*
  • Proteins / isolation & purification
  • Proteins / metabolism
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Restriction Mapping
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins
  • Sequence Deletion
  • Sequence Homology, Amino Acid
  • beta-Galactosidase / genetics
  • beta-Galactosidase / metabolism

Substances

  • Cell Cycle Proteins
  • Fungal Proteins
  • Histones
  • NAP1 protein, S cerevisiae
  • Nap1l1 protein, mouse
  • Nuclear Proteins
  • Nucleosome Assembly Protein 1
  • Proteins
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • beta-Galactosidase