Selective immunoneutralization of the multiple activities of Escherichia coli DNA polymerase I supports the model for separate active sites and indicates a complex 5' to 3' exonuclease.
DNA polymerase I (pol I) from Escherichia coli has three well-defined activities: DNA polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. We have raised monoclonal antibodies to pol I which selectively neutralize each of these three activities, thus supporting the model of separate active sites for each activity, heretofore exclusively demonstrated with proteolytic fragments of pol I. Antibodies from each class could bind pol I in the presence of antibodies of another class, indicating the existence of significant spatial separation between each of the three sites. In addition, several of the neutralizing antibodies were able to distinguish particular activities of the 5'-3' exonuclease. One of them, for example, inhibited the RNase H activity but not the DNase activity. Two other antibodies could, in addition to inhibiting the polymerase and the 3'-5' exonuclease, either stimulate or inhibit the 5'-3' exonuclease depending upon the assay conditions, particularly the ionic strength.
Ruscitti
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Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
Polayes
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DA
Karu
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AE
Linn
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S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D000936:Antigen-Antibody Complex; D001665:Binding Sites; D004265:DNA Helicases / Q000378:metabolism*; D004256:DNA Polymerase I / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008815:Mice, Inbred Strains / Q000276:immunology; D009500:Neutralization Tests; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D010446:Peptide Fragments; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D004265:DNA Helicases