title: Nucleosome assembly and disassembly activity of GRWD1, a novel Cdt1-binding protein that promotes pre-replication complex formation.
abstract: GRWD1 was previously identified as a novel Cdt1-binding protein that possesses histone-binding and nucleosome assembly activities and promotes MCM loading, probably by maintaining chromatin openness at replication origins. However, the molecular mechanisms underlying these activities remain unknown. We prepared reconstituted mononucleosomes from recombinant histones and a DNA fragment containing a nucleosome positioning sequence, and investigated the effects of GRWD1 on them. GRWD1 could disassemble these preformed mononucleosomes in vitro in an ATP-independent manner. Thus, our data suggest that GRWD1 facilitates removal of H2A-H2B dimers from nucleosomes, resulting in formation of hexasomes. The activity was compromised by deletion of the acidic domain, which is required for efficient histone binding. In contrast, nucleosome assembly activity of GRWD1 was not affected by deletion of the acidic domain. In HeLa cells, the acidic domain of GRWD1 was necessary to maintain chromatin openness and promote MCM loading at replication origins. Taken together, our results suggest that GRWD1 promotes chromatin fluidity by influencing nucleosome structures, e.g., by transient eviction of H2A-H2B, and thereby promotes efficient MCM loading at replication origins.
authors.affiliation: Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Aizawa
authors.affiliation: Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Sugimoto
authors.affiliation: Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan.
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Yoshida
authors.affiliation: Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582, Japan. Electronic address: mfujita@phar.kyushu-u.ac.jp.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Fujita
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D042002:Chromatin Assembly and Disassembly; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D064110:Minichromosome Maintenance Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D034622:RNA Interference; D018741:Replication Origin; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: C467199:CDT1 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C504316:GRWD1 protein, human; D006657:Histones; D009707:Nucleosomes; D004247:DNA; D064110:Minichromosome Maintenance Proteins
keywords: Cdt1; Chromatin; DNA replication; GRWD1; Histone chaperone; MCM