ABSTRACT
An in situ technique for studying the chromatin binding of proteins in single fission yeast cells (Schizosaccharomyces pombe) is described. Cells are permeabilized by enzymatic digestion and extracted with a detergent-containing buffer. This procedure removes soluble proteins, but proteins that are bound to insoluble cell structures such as chromatin are retained, and overall cell morphology is maintained. Extraction of proteins is monitored by fluorescence microscopy, either using fluorescently tagged proteins or by indirect immunofluorescence. This method allows the chromatin association of proteins to be correlated with other cell cycle events without the need for cell synchronization.
Subject(s)
Chromatin/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Amino Acid Sequence , Base Sequence , Chromatin/genetics , DNA, Fungal/genetics , DNA, Recombinant/genetics , Detergents , Flow Cytometry , Genetic Vectors , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Micrococcal Nuclease , Molecular Biology/methods , Molecular Sequence Data , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/geneticsABSTRACT
Using a cytological assay to monitor the successive chromatin association of replication proteins leading to replication initiation, we have investigated the function of fission yeast Cdc23/Mcm10 in DNA replication. Inactivation of Cdc23 before replication initiation using tight degron mutations has no effect on Mcm2 chromatin association, and thus pre-replicative complex (pre-RC) formation, although Cdc45 chromatin binding is blocked. Inactivating Cdc23 during an S phase block after Cdc45 has bound causes a small reduction in Cdc45 chromatin binding, and replication does not terminate in the absence of Mcm10 function. These observations show that Cdc23/Mcm10 function is conserved between fission yeast and Xenopus, where in vitro analysis has indicated a similar requirement for Cdc45 binding, but apparently not compared with Saccharomyces cerevisiae, where Mcm10 is needed for Mcm2 chromatin binding. However, unlike the situation in Xenopus, where Mcm10 chromatin binding is dependent on Mcm2-7, we show that the fission yeast protein is bound to chromatin throughout the cell cycle in growing cells, and only displaced from chromatin during quiescence. On return to growth, Cdc23 chromatin binding is rapidly reestablished independently from pre-RC formation, suggesting that chromatin association of Cdc23 provides a link between proliferation and competence to execute DNA replication.
