Abstract
Eukaryotes use distinct polymerases for leading- and lagging-strand replication, but how they target their respective strands is uncertain. We reconstituted Saccharomyces cerevisiae replication forks and found that CMG helicase selects polymerase (Pol) ɛ to the exclusion of Pol δ on the leading strand. Even if Pol δ assembles on the leading strand, Pol ɛ rapidly replaces it. Pol δ-PCNA is distributive with CMG, in contrast to its high stability on primed ssDNA. Hence CMG will not stabilize Pol δ, instead leaving the leading strand accessible for Pol ɛ and stabilizing Pol ɛ. Comparison of Pol ɛ and Pol δ on a lagging-strand model DNA reveals the opposite. Pol δ dominates over excess Pol ɛ on PCNA-primed ssDNA. Thus, PCNA strongly favors Pol δ over Pol ɛ on the lagging strand, but CMG over-rides and flips this balance in favor of Pol ɛ on the leading strand.
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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DNA Polymerase II / chemistry*
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DNA Polymerase III / chemistry*
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DNA Replication*
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DNA, Fungal / biosynthesis
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DNA, Fungal / chemistry
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DNA, Fungal / genetics
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DNA-Binding Proteins / chemistry
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Kinetics
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Minichromosome Maintenance Proteins / chemistry
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Nuclear Proteins / chemistry
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Proliferating Cell Nuclear Antigen / chemistry
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Replication Protein A / chemistry
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Ribonucleoprotein, U4-U6 Small Nuclear / chemistry
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Ribonucleoprotein, U5 Small Nuclear / chemistry
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Saccharomyces cerevisiae / enzymology*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae Proteins / chemistry
Substances
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CDC45 protein, S cerevisiae
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DNA, Fungal
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DNA-Binding Proteins
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Nuclear Proteins
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POL30 protein, S cerevisiae
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PRP8 protein, S cerevisiae
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Proliferating Cell Nuclear Antigen
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Replication Protein A
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Ribonucleoprotein, U4-U6 Small Nuclear
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Ribonucleoprotein, U5 Small Nuclear
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Saccharomyces cerevisiae Proteins
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DNA Polymerase II
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DNA Polymerase III
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Minichromosome Maintenance Proteins