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

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.

[1]  Anindya Dutta,et al.  DNA replication in eukaryotic cells. , 2002, Annual review of biochemistry.

[2]  S. Forsburg,et al.  Nuclear localization of Schizosaccharomyces pombe Mcm2/Cdc19p requires MCM complex assembly. , 1999, Molecular biology of the cell.

[3]  P. Nurse,et al.  Mis-specification of cortical identity in a fission yeast PAK mutant , 1999, Current Biology.

[4]  P. Nurse,et al.  The fission yeast origin recognition complex is constitutively associated with chromatin and is differentially modified through the cell cycle , 1999 .

[5]  Stephen E. Kearsey,et al.  G1-phase and B-type cyclins exclude the DNA-replication factor Mcm4 from the nucleus , 1999, Nature Cell Biology.

[6]  J. Hurwitz,et al.  Identification and reconstitution of the origin recognition complex from Schizosaccharomyces pombe. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Tatsuro S. Takahashi,et al.  Association of Fission Yeast Orp1 and Mcm6 Proteins with Chromosomal Replication Origins , 1999, Molecular and Cellular Biology.

[8]  H. Masukata,et al.  Clustered Adenine/Thymine Stretches Are Essential for Function of a Fission Yeast Replication Origin , 1999, Molecular and Cellular Biology.

[9]  K. Nasmyth,et al.  Whose end is destruction: cell division and the anaphase-promoting complex. , 1999, Genes & development.

[10]  R. Chuang,et al.  The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[11]  K. Nasmyth.  Separating sister chromatids. , 1999, Trends in biochemical sciences.

[12]  B. Tye.  MCM proteins in DNA replication. , 1999, Annual review of biochemistry.

[13]  P. Nurse,et al.  Cdc18 transcription and proteolysis couple S phase to passage through mitosis , 1998, The EMBO journal.

[14]  M. Méchali,et al.  Dynamics of the Genome during Early Xenopus laevis Development: Karyomeres As Independent Units of Replication , 1998, The Journal of cell biology.

[15]  P. Plevani,et al.  Evidence for a Cdc6p‐independent mitotic resetting event involving DNA polymerase α , 1998, The EMBO journal.

[16]  S. Kearsey,et al.  MCM proteins: evolution, properties, and role in DNA replication. , 1998, Biochimica et biophysica acta.

[17]  M. Yanagida.  Fission yeast cut mutations revisited: control of anaphase. , 1998, Trends in cell biology.

[18]  J. Li,et al.  Ectopic induction of Clb2 in early G1 phase is sufficient to block prereplicative complex formation in Saccharomyces cerevisiae. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[19]  P. Russell,et al.  Negative regulation of Cdc18 DNA replication protein by Cdc2. , 1998, Molecular biology of the cell.

[20]  B. Stillman,et al.  Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants. , 1997, Genes & development.

[21]  P. Jallepalli,et al.  Regulation of the replication initiator protein p65cdc18 by CDK phosphorylation. , 1997, Genes & development.

[22]  S. Prinz,et al.  CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. , 1997, Science.

[23]  O. Aparicio,et al.  Components and Dynamics of DNA Replication Complexes in S. cerevisiae: Redistribution of MCM Proteins and Cdc45p during S Phase , 1997, Cell.

[24]  Y. Ishimi.  A DNA Helicase Activity Is Associated with an MCM4, -6, and -7 Protein Complex* , 1997, The Journal of Biological Chemistry.

[25]  K. Nasmyth,et al.  Loading of an Mcm Protein onto DNA Replication Origins Is Regulated by Cdc6p and CDKs , 1997, Cell.

[26]  M. Yanagida,et al.  A globular complex formation by Nda1 and the other five members of the MCM protein family in fission yeast , 1997, Genes to cells : devoted to molecular & cellular mechanisms.

[27]  Y. Endo,et al.  HsMCM6: a new member of the human MCM/P1 family encodes a protein homologous to fission yeast Mis5 , 1997, Genes to cells : devoted to molecular & cellular mechanisms.

[28]  L. Drury,et al.  Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Jim Haseloff,et al.  Mutations that suppress the thermosensitivity of green fluorescent protein , 1996, Current Biology.

[30]  J. Diffley,et al.  Once and only once upon a time: specifying and regulating origins of DNA replication in eukaryotic cells. , 1996, Genes & development.

[31]  J. Blow,et al.  The Xenopus origin recognition complex is essential for DNA replication and MCM binding to chromatin , 1996, Current Biology.

[32]  G. Evan,et al.  Interaction between the Origin Recognition Complex and the Replication Licensing Systemin Xenopus , 1996, Cell.

[33]  P. Nurse,et al.  The ORC1 homolog orp1 in fission yeast plays a key role in regulating onset of S phase. , 1996, Genes & development.

[34]  T. Coleman,et al.  The Xenopus Cdc6 Protein Is Essential for the Initiation of a Single Round of DNA Replication in Cell-Free Extracts , 1996, Cell.

[35]  T. Böhm,et al.  Activation of S-phase-promoting CDKs in late G1 defines a "point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast. , 1996, Genes & development.

[36]  I. Todorov,et al.  Large, complex modular structure of a fission yeast DNA replication origin , 1996, Current Biology.

[37]  T. Kelly,et al.  cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[38]  Greet Blom van Assendelft,et al.  Fission yeast cdc21, a member of the MCM protein family, is required for onset of S phase and is located in the nucleus throughout the cell cycle. , 1996, The EMBO journal.

[39]  K. Nasmyth,et al.  An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast , 1996, Nature.

[40]  G. Pierron,et al.  34 DNA Replication in Physarum , 1996 .

[41]  T. Kelly,et al.  Genetic analysis of an ARS element from the fission yeast Schizosaccharomyces pombe. , 1995, The EMBO journal.

[42]  P. Nurse,et al.  p65 cdc18 Plays a major role controlling the initiation of DNA replication in fission yeast , 1995, Cell.

[43]  K. Nasmyth,et al.  S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state , 1995, Current Biology.

[44]  A. Attaran,et al.  BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication , 1995, The Journal of cell biology.

[45]  H. Nojima,et al.  Identification of the yeast MCM3-related protein as a component of xenopus DNA replication licensing factor , 1995, Cell.

[46]  Kim Nasmyth,et al.  Genes involved in sister chromatid separation are needed for b-type cyclin proteolysis in budding yeast , 1995, Cell.

[47]  S. Dalton,et al.  Cell cycle-regulated nuclear import and export of Cdc47, a protein essential for initiation of DNA replication in budding yeast. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[48]  R. Fotedar,et al.  Cell cycle control of DNA replication. , 1995, Progress in cell cycle research.

[49]  M. Yanagida,et al.  Fission yeast minichromosome loss mutants mis cause lethal aneuploidy and replication abnormality. , 1994, Molecular biology of the cell.

[50]  J. Diffley,et al.  Two steps in the assembly of complexes at yeast replication origins in vivo , 1994, Cell.

[51]  G. Lucchini,et al.  The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication , 1994, Molecular and cellular biology.

[52]  B. Tye,et al.  Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast. , 1993, Genes & development.

[53]  M. Yanagida,et al.  Visualization of centromeric and nucleolar DNA in fission yeast by fluorescence in situ hybridization. , 1992, Journal of cell science.

[54]  S. Moreno,et al.  Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. , 1991, Methods in enzymology.

[55]  D. Botstein,et al.  Subcellular localization of yeast CDC46 varies with the cell cycle. , 1990, Genes & development.

[56]  D. Beach,et al.  The fission yeast cdc2/cdc13/suc1 protein kinase: Regulation of catalytic activity and nuclear localization , 1989, Cell.

[57]  Sergio Moreno,et al.  Regulation of p34cdc2 protein kinase during mitosis , 1989, Cell.

[58]  K. Gull,et al.  Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. , 1989, Journal of cell science.

[59]  T. Toda,et al.  Sequential alterations in the nuclear chromatin region during mitosis of the fission yeast Schizosaccharomyces pombe: video fluorescence microscopy of synchronously growing wild-type and cold-sensitive cdc mutants by using a DNA-binding fluorescent probe. , 1981, Journal of cell science.