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Nucleic Acids Res. 2014 Oct;42(18):11657-67. doi: 10.1093/nar/gku785. Epub 2014 Sep 17.

Single-molecule analysis uncovers the difference between the kinetics of DNA decatenation by bacterial topoisomerases I and III.

Author information

1
Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA.
2
Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA Department of Physics and Astronomy, Northwestern University, Evanston, IL 60208, USA.
3
Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208, USA a-mondragon@northwestern.edu.

Abstract

Escherichia coli topoisomerases I and III can decatenate double-stranded DNA (dsDNA) molecules containing single-stranded DNA regions or nicks as well as relax negatively supercoiled DNA. Although the proteins share a mechanism of action and have similar structures, they participate in different cellular processes. Whereas topoisomerase III is a more efficient decatenase than topoisomerase I, the opposite is true for DNA relaxation. In order to investigate the differences in the mechanism of these two prototypical type IA topoisomerases, we studied DNA decatenation at the single-molecule level using braids of intact dsDNA and nicked dsDNA with bulges. We found that neither protein decatenates an intact DNA braid. In contrast, both enzymes exhibited robust decatenation activity on DNA braids with a bulge. The experiments reveal that a main difference between the unbraiding mechanisms of these topoisomerases lies in the pauses between decatenation cycles. Shorter pauses for topoisomerase III result in a higher decatenation rate. In addition, topoisomerase III shows a strong dependence on the crossover angle of the DNA strands. These real-time observations reveal the kinetic characteristics of the decatenation mechanism and help explain the differences between their activities.

PMID:
25232096
PMCID:
PMC4191389
DOI:
10.1093/nar/gku785
[Indexed for MEDLINE]
Free PMC Article

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