Decatenating activity of Escherichia coli DNA gyrase and topoisomerases I and III during oriC and pBR322 DNA replication in vitro

• Published in: The Journal of biological chemistry Vol. 269; no. 3; pp. 2093 - 2099
• Main Authors: HIASA, H, DIGATE, R. J, MARIANS, K. J
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 21-01-1994
• Subjects: Adenosine Triphosphate - metabolism; Biological and medical sciences; Chromatography, Affinity; Chromatography, DEAE-Cellulose; DNA Replication; DNA Topoisomerases, Type I - isolation & purification; DNA Topoisomerases, Type I - metabolism; DNA Topoisomerases, Type II - isolation & purification; DNA Topoisomerases, Type II - metabolism; DNA, Bacterial - biosynthesis; DNA, Circular - biosynthesis; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Kinetics; Molecular and cellular biology; Molecular genetics; Plasmids; Replication; Plasmid; DNA topoisomerase (ATP-hydrolysing); Isomerases; Enzymatic activity; Radiolabelling; Enzyme; Gel electrophoresis; Escherichia coli; DNA topoisomerase; Bacteria; Replication; Enterobacteriaceae
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)42140-5

oriC and pBR322 DNA replication, reconstituted with purified replication proteins, has been used to study the functional activities
of Escherichia coli topoisomerase I, DNA gyrase, and topoisomerase III during the final stages of DNA replication. In the
oriC system, DNA gyrase-catalyzed decatenation of daughter DNA molecules was very inefficient, whereas topoisomerase III could
catalyze complete decatenation. In the pBR322 DNA replication system, almost all the daughter DNA molecules could be decatenated
by DNA gyrase alone in the absence of salt. Decatenation by DNA gyrase in the pBR322 system was completely inhibited, without
a concomitant inhibition of DNA synthesis, by the addition of physiological concentrations of salt. Topoisomerase III, however,
could decatenate all of the daughter DNA molecules in the pBR322 system, even in the presence of high concentrations of salt.
A similar effect could not be observed in the oriC system, because the addition of salt inhibited DNA synthesis. Topoisomerase
I was incapable of catalyzing decatenation under any conditions examined in either the oriC or pBR322 replication system.
The addition of topoisomerase I to the replication systems resulted only in an inhibition of DNA synthesis.