Interaction of DNA polymerase delta, proliferating cell nuclear antigen, and synthetic oligonucleotide template-primers. Analysis by polyacrylamide gel electrophoresis-band mobility shift assay.
A polyacrylamide gel electrophoresis band-mobility shift assay was developed to study the binding of synthetic oligonucleotides by DNA polymerase delta (pol delta) and proliferating cell nuclear antigen (PCNA). As measured by this assay, neither calf thymus pol delta core enzyme nor PCNA alone bind DNA stably. However, mammalian PCNA but not Drosophila PCNA promotes the formation of a distinct pol delta.PCNA.template-primer complex. Appearance of this complex is primer-dependent but does not require Mg2+. Complex stability is also influenced by the presence or absence of individual dNTPs. A model for the ordered sequential interaction of pol delta, PCNA, and DNA template-primers is proposed.
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Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651.
McConnell
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Tan
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Downey
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Fisher
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D004258:DNA Polymerase III; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen; D013698:Templates, Genetic; D013950:Thymus Gland / Q000201:enzymology
D004277:DNA, Single-Stranded; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium