title: Imaging analysis to determine chromatin binding of the licensing factor MCM2-7 in mammalian cells.
abstract: S-CDK and DDK protein kinases initiate DNA replication at replication origins. Prior to the activation of these kinases, origins must become competent for replication by loading MCM2-7 DNA helicase on chromatin. This process is known as replication licensing or pre-replicative complex (pre-RC) formation. After the onset of S phase, however, licensing is inhibited to prevent re-replication of DNA. In this chapter, we describe a method to analyze origin licensing by imaging the chromatin-bound licensing factor MCM2-7. In a normal cell cycle, MCM2-7 is loaded at the end of mitosis or early G1 phase. As S phase progresses, MCM2-7 is dissociated from the replicated regions. When DNA replication is completed, cells in G2 phase have no chromatin-bound MCM2-7. The analysis of chromatin-bound MCM2-7 in each cell provides an insight into cell cycle stage and condition for cell cycle.
authors.affiliation: Graduate School of Life Science, University of Hyogo, Kamigori, Ako-gun, Hyogo, 678-1297, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Morino
authors.forename: Miyuki
authors.initials: M
authors.lastname: Tanaka
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Shiomi
authors.forename: Hideo
authors.initials: H
authors.lastname: Nishitani
mesh_terms: D002453:Cell Cycle; D002843:Chromatin / Q000378:metabolism*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D064110:Minichromosome Maintenance Proteins / Q000032:analysis / Q000378:metabolism*; D061848:Optical Imaging / Q000379:methods; D011485:Protein Binding; D016707:Tissue Fixation / Q000379:methods
chemical_list: D002843:Chromatin; D064110:Minichromosome Maintenance Proteins