Analysis of Mcm2-7 chromatin binding during anaphase and in the transition to quiescence in fission yeast.
Mcm2-7 proteins are generally considered to function as a heterohexameric complex, providing helicase activity for the elongation step of DNA replication. These proteins are loaded onto replication origins in M-G1 phase in a process termed licensing or pre-replicative complex formation. It is likely that Mcm2-7 proteins are loaded onto chromatin simultaneously as a pre-formed hexamer although some studies suggest that subcomplexes are recruited sequentially. To analyze this process in fission yeast, we have compared the levels and chromatin binding of Mcm2-7 proteins during the fission yeast cell cycle. Mcm subunits are present at approximately 1 x 10(4) molecules/cell and are bound with approximately equal stoichiometry on chromatin in G1/S phase cells. Using a single cell assay, we have correlated the timing of chromatin association of individual Mcm subunits with progression through mitosis. This showed that Mcm2, 4 and 7 associate with chromatin at about the same stage of anaphase, suggesting that licensing involves the simultaneous binding of these subunits. We also examined Mcm2-7 chromatin association when cells enter a G0-like quiescent state. Chromatin binding is lost in this transition in a process that does not require DNA replication or the selective degradation of specific subunits.
Namdar
Mandana
M
Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS, UK.
Kearsey
Stephen E
SE
D000705:Anaphase; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000032:analysis / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; D012568:Schizosaccharomyces / Q000166:cytology* / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000032:analysis / Q000378:metabolism*
D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D029702:Schizosaccharomyces pombe Proteins; C085711:mcm5 protein, S pombe; D049452:Green Fluorescent Proteins; C514502:Mcm7 protein, S pombe; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; C085710:mcm2 protein, S pombe; C078490:mcm4 protein, S pombe; C092300:mcm6 protein, S pombe