Abstract
We have reconstituted a eukaryotic leading/lagging strand replisome comprising 31 distinct polypeptides. This study identifies a process unprecedented in bacterial replisomes. While bacteria and phage simply recruit polymerases to the fork, we find that suppression mechanisms are used to position the distinct eukaryotic polymerases on their respective strands. Hence, Pol ε is active with CMG on the leading strand, but it is unable to function on the lagging strand, even when Pol δ is not present. Conversely, Pol δ-PCNA is the only enzyme capable of extending Okazaki fragments in the presence of Pols ε and α. We have shown earlier that Pol δ-PCNA is suppressed on the leading strand with CMG (Georgescu et al., 2014). We propose that CMG, the 11-subunit helicase, is responsible for one or both of these suppression mechanisms that spatially control polymerase occupancy at the fork.
Keywords:
CMG; DNA replication; Pol delta; Pol epsilon; S. cerevisiae; biochemistry; biophysics; replication fork; structural biology.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Base Sequence
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DNA / genetics
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DNA / metabolism
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DNA Helicases / chemistry
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DNA Helicases / genetics*
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DNA Helicases / metabolism
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DNA Polymerase I / genetics
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DNA Polymerase I / metabolism
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DNA Polymerase II / genetics
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DNA Polymerase II / metabolism
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DNA Polymerase beta / genetics
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DNA Polymerase beta / metabolism
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DNA Replication*
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DNA, Fungal / chemistry
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DNA, Fungal / genetics*
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DNA, Fungal / metabolism
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Gene Expression
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Molecular Sequence Data
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Protein Subunits / chemistry
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Protein Subunits / genetics*
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Protein Subunits / metabolism
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae / genetics*
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / genetics*
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Saccharomyces cerevisiae Proteins / metabolism
Substances
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DNA, Fungal
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Okazaki fragments
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Protein Subunits
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins
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DNA
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DNA Polymerase I
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DNA Polymerase II
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DNA Polymerase beta
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DNA Helicases