Chromatin binding of the fission yeast replication factor mcm4 occurs during anaphase and requires ORC and cdc18

AUTOR(ES)

Kearsey, Stephen E.

FONTE

Oxford University Press

RESUMO

We describe an in situ technique for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe. After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be determined from individual cells in asynchronous cultures. We have applied this method to the DNA replication factor mcm4/cdc21, and find that chromatin association occurs during anaphase B, significantly earlier than is the case in budding yeast. Binding of mcm4 to chromatin requires orc1 and cdc18 (homologous to Cdc6 in budding yeast). Release of mcm4 from chromatin occurs during S phase and requires DNA replication. Upon overexpressing cdc18, we show that mcm4 is required for re-replication of the genome in the absence of mitosis and is associated with chromatin in cells undergoing re-replication.

Documentos Relacionados

• Interaction of the S phase regulator Cdc18 with cyclin-dependent kinase in fission yeast
• Fission Yeast Cdc23/Mcm10 Functions after Pre-replicative Complex Formation To Promote Cdc45 Chromatin Binding
• Negative Regulation of Cdc18 DNA Replication Protein by Cdc2
• Multiple ORC-binding sites are required for efficient MCM loading and origin firing in fission yeast
• sud1+ targets cyclin-dependent kinase-phosphorylated Cdc18 and Rum1 proteins for degradation and stops unwanted diploidization in fission yeast