Abstract
Replication protein A (RPA) is an essential component of DNA metabolic processes. RPA binds to single-stranded DNA (ssDNA) and interacts with multiple DNA-binding proteins. In this study, we showed that two DNA polymerases, PolB and PolD, from the hyperthermophilic archaeon Thermococcus kodakarensis interact directly with RPA in vitro. RPA was expected to play a role in resolving the secondary structure, which may stop the DNA synthesis reaction, in the template ssDNA. Our in vitro DNA synthesis assay showed that the pausing was resolved by RPA for both PolB and PolD. These results supported the fact that RPA interacts with DNA polymerases as a member of the replisome and is involved in the normal progression of DNA replication forks.
Keywords:
Archaea; DNA polymerase; DNA replication; RPA; replication fork progression.
MeSH terms
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Archaeal Proteins / genetics*
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Archaeal Proteins / metabolism
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Cloning, Molecular
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DNA Replication*
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DNA, Archaeal / genetics
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DNA, Archaeal / metabolism
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DNA, Single-Stranded / genetics
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DNA, Single-Stranded / metabolism
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DNA-Directed DNA Polymerase / genetics*
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DNA-Directed DNA Polymerase / metabolism
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Gene Expression
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Gene Expression Regulation, Archaeal*
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Genetic Vectors / chemistry
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Genetic Vectors / metabolism
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Isoenzymes / genetics
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Isoenzymes / metabolism
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Protein Binding
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Replication Protein A / genetics*
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Replication Protein A / metabolism
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Thermococcus / genetics*
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Thermococcus / metabolism
Substances
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Archaeal Proteins
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DNA, Archaeal
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DNA, Single-Stranded
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Isoenzymes
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Recombinant Proteins
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Replication Protein A
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DNA-Directed DNA Polymerase