Abstract
Leading-strand template aberrations cause helicase-polymerase uncoupling and impede replication fork progression, but the details of how uncoupled forks are restarted remain uncertain. Using purified proteins from Saccharomyces cerevisiae, we have reconstituted translesion synthesis (TLS)-mediated restart of a eukaryotic replisome following collision with a cyclobutane pyrimidine dimer. We find that TLS functions 'on the fly' to promote resumption of rapid replication fork rates, despite lesion bypass occurring uncoupled from the Cdc45-MCM-GINS (CMG) helicase. Surprisingly, the main lagging-strand polymerase, Pol δ, binds the leading strand upon uncoupling and inhibits TLS. Pol δ is also crucial for efficient recoupling of leading-strand synthesis to CMG following lesion bypass. Proliferating cell nuclear antigen monoubiquitination positively regulates TLS to overcome Pol δ inhibition. We reveal that these mechanisms of negative and positive regulation also operate on the lagging strand. Our observations have implications for both fork restart and the division of labor during leading-strand synthesis generally.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Acetyltransferases / genetics
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Acetyltransferases / metabolism
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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DNA Polymerase II / genetics
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DNA Polymerase II / metabolism
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DNA Polymerase III / genetics
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DNA Polymerase III / metabolism
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DNA Repair
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DNA Replication*
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Minichromosome Maintenance Proteins / genetics
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Minichromosome Maintenance Proteins / metabolism
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Proliferating Cell Nuclear Antigen / genetics
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Proliferating Cell Nuclear Antigen / 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*
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Ubiquitin-Activating Enzymes / genetics
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Ubiquitin-Activating Enzymes / metabolism
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Ubiquitination
Substances
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CDC45 protein, S cerevisiae
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Cell Cycle Proteins
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DNA-Binding Proteins
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MRC1 protein, S cerevisiae
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Membrane Proteins
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Nuclear Proteins
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POL30 protein, S cerevisiae
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Proliferating Cell Nuclear Antigen
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Saccharomyces cerevisiae Proteins
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Acetyltransferases
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ELO2 protein, S cerevisiae
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DNA Polymerase II
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DNA Polymerase III
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Minichromosome Maintenance Proteins
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Uba1 protein, S cerevisiae
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Ubiquitin-Activating Enzymes