Abstract
By using a complementation assay for a replication factor C dependent DNA polymerase activity on a singly-primed M13 DNA template, we have isolated from calf thymus a multiprotein complex active in DNA replication. For this, the inclusion of ATP during the entire isolation procedure was essential, since the complex decayed after omission of ATP. This complex contains at least DNA polymerase alpha/primase, DNA polymerase delta, and replication factor C as shown by gel-filtration and coimmunoprecipitation experiments. It is functionally active in replication of primed and unprimed single-stranded M13 DNA templates. Furthermore, in the presence of proliferating cell nuclear antigen and ATP, it forms an isolatable holoenzyme/template-primer complex. Replication factor C apparently mediates the interaction of DNA polymerase delta in the complex with proliferating cell nuclear antigen, through an ATP-dependent mechanism. This interaction appears to stabilize the binding of the complex to a template-primer and to coordinate the activity of DNA polymerase alpha/primase and DNA polymerase delta during replication of a single-stranded DNA template. Our data suggest the existence of an asymmetric DNA polymerase complex in mammalian cells.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphate / metabolism
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Animals
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Antibodies, Monoclonal
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Cattle
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Chromatography, Gel
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DNA Polymerase II / chemistry
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DNA Polymerase II / isolation & purification
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DNA Polymerase II / metabolism*
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DNA Polymerase III
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DNA Primase
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DNA Primers / metabolism
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DNA Replication / physiology*
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DNA, Single-Stranded / metabolism
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DNA-Binding Proteins / chemistry
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DNA-Binding Proteins / isolation & purification
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DNA-Binding Proteins / metabolism*
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DNA-Directed DNA Polymerase / chemistry
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DNA-Directed DNA Polymerase / isolation & purification
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DNA-Directed DNA Polymerase / metabolism*
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Enzyme Stability
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Homeodomain Proteins*
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Humans
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In Vitro Techniques
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Kinetics
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Minor Histocompatibility Antigens
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Precipitin Tests
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Proliferating Cell Nuclear Antigen / metabolism
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Protein Binding
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Protein Conformation
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Proto-Oncogene Proteins c-bcl-2*
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RNA Nucleotidyltransferases / metabolism
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Replication Protein C
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Repressor Proteins*
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Saccharomyces cerevisiae Proteins*
Substances
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Antibodies, Monoclonal
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BCL2-related protein A1
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DNA Primers
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DNA, Single-Stranded
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DNA-Binding Proteins
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Homeodomain Proteins
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MATA1 protein, S cerevisiae
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Minor Histocompatibility Antigens
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Proliferating Cell Nuclear Antigen
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Proto-Oncogene Proteins c-bcl-2
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Repressor Proteins
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Saccharomyces cerevisiae Proteins
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Adenosine Triphosphate
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DNA Primase
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RNA Nucleotidyltransferases
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DNA Polymerase II
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DNA Polymerase III
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DNA-Directed DNA Polymerase
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Replication Protein C