Abstract
Although the molecular enzymology of DNA replication is well characterised, how and why it occurs in discrete nuclear foci is unclear. Using fission yeast, we show that replication takes place in a limited number of replication foci, whose distribution changes with progression through S phase. These sites define replication factories which contain on average 14 replication forks. We show for the first time that entire foci are mobile, able both to fuse and re-segregate. These foci form distinguishable patterns during S phase, whose succession is reproducible, defining early-, mid- and late-S phase. In wild-type cells, this same temporal sequence can be detected in the presence of hydroxyurea (HU), despite the reduced rate of replication. In cells lacking the intra-S checkpoint kinase Cds1, replication factories dismantle on HU. Intriguingly, even in the absence of DNA damage, the replication foci in cds1 cells assume a novel distribution that is not present in wild-type cells, arguing that Cds1 kinase activity contributes to the spatio-temporal organisation of replication during normal cell growth.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Cell Cycle Proteins / genetics*
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Cell Cycle Proteins / metabolism
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Checkpoint Kinase 1
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DNA Helicases / genetics
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DNA Helicases / metabolism
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DNA Replication / drug effects
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DNA Replication / genetics*
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DNA Replication / physiology
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism
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Flow Cytometry
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism
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Hydroxyurea / pharmacology
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Kinetics
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Origin Recognition Complex / genetics
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Proliferating Cell Nuclear Antigen / genetics
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Proliferating Cell Nuclear Antigen / metabolism
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Protein Kinases / genetics
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Protein Kinases / metabolism
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism
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S Phase / drug effects
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S Phase / genetics*
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Schizosaccharomyces / drug effects
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Schizosaccharomyces / genetics*
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Schizosaccharomyces / metabolism
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Schizosaccharomyces pombe Proteins / genetics
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Schizosaccharomyces pombe Proteins / metabolism
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Time Factors
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Trans-Activators / genetics
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Trans-Activators / metabolism
Substances
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Cell Cycle Proteins
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DNA-Binding Proteins
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Nuclear Proteins
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Origin Recognition Complex
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Proliferating Cell Nuclear Antigen
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Schizosaccharomyces pombe Proteins
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Trans-Activators
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replication initiator protein
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Green Fluorescent Proteins
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Protein Kinases
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Checkpoint Kinase 1
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Protein Serine-Threonine Kinases
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DNA Helicases
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Hydroxyurea