Functional analysis of nucleosome assembly protein, NAP-1. The negatively charged COOH-terminal region is not necessary for the intrinsic assembly activity.
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.
Fujii-Nakata
T
T
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Ishimi
Y
Y
Okuda
A
A
Kikuchi
A
A
D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D018797:Cell Cycle Proteins; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005800:Genes, Fungal; D006657:Histones / Q000378:metabolism; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D056490:Nucleosome Assembly Protein 1; D010957:Plasmids; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D006657:Histones; C533556:NAP1 protein, S cerevisiae; C533558:Nap1l1 protein, mouse; D009687:Nuclear Proteins; D056490:Nucleosome Assembly Protein 1; D011506:Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D001616:beta-Galactosidase