RRC ID 57704
Author Fujii-Nakata T, Ishimi Y, Okuda A, Kikuchi A.
Title Functional analysis of nucleosome assembly protein, NAP-1. The negatively charged COOH-terminal region is not necessary for the intrinsic assembly activity.
Journal J Biol Chem
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.
Volume 267(29)
Pages 20980-6
Published 1992-10-15
PMID 1400414
MeSH 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
IF 4.106
Times Cited 130
Resource
DNA material Yeast NAP-1 (RDB01356)