Relaxation complexes of poasmid DNA and protein. III. Association of protein with the 5' terminus of the broken DNA strand in the relaxed complex of plasmid ColE1.
The location of the protein in the open circular DNA form of the ColE1 DNA-protein relaxation complex, induced by treatment with sodium dodecyl sulfate, has been studied using several enzymes of DNA metabolism. Escherichia coli exonucleases I and III are able to degrade extensively the nicked strand of the relaxed complex from the 3' end. DNA polymerase I can initiate synthesis using the relaxed complex as template-primer and specifically extends the 3' end of the nicked strand. The 5' end of the sodium dodecyl sulfate-relaxed complex, however, is blocked to the 5'-3' hydrolitic action T7 exonuclease. This block remains after trypsin treatment of the sodium dodecyl sulfate-relaxed complex but is removed by Pronase treatment. T4 DNA ligase is unable to seal either the sodium dodecyl sulfate-relaxed complex or the Pronase-treated relaxed complex even after pretreatment of the relaxed complex with T4 DNA polymerase and polynucleotide kinase. However, pretreatment with DNA polymerase I and the four deoxyribonucleoside triphosphates facilitates ligase closure of the Pronase-treated relaxed complex but not the sodium dodecyl sulfate-relaxed complex. These studies indicate that the protein in the relaxed ColE1 complex is located at or near the 5' end of the nicked strand.
D001426:Bacterial Proteins; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D004254:DNA Nucleotidyltransferases; D004269:DNA, Bacterial; D004270:DNA, Circular; D003851:Deoxyribonucleases; D004926:Escherichia coli / Q000032:analysis* / Q000201:enzymology; D005092:Exonucleases; D046911:Macromolecular Substances; D009690:Nucleic Acid Conformation; D009876:Operon; D011118:Polynucleotide Ligases; D011402:Pronase; D011485:Protein Binding; D011487:Protein Conformation; D014357:Trypsin
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004270:DNA, Circular; D046911:Macromolecular Substances; D004254:DNA Nucleotidyltransferases; D003851:Deoxyribonucleases; D005092:Exonucleases; D014357:Trypsin; D011402:Pronase; D011118:Polynucleotide Ligases