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J. Biol. Chem., Vol. 281, Issue 35, 25407-25415, September 1, 2006

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Topoisomerase Action on Short DNA Duplexes Reveals Requirements for Gate and Transfer DNA Segments^*

Boris P. Belotserkovskii, Paola B. Arimondo¹, and Nicholas R. Cozzarelli

From the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3402

Type II topoisomerases change DNA topology by passage of one DNA duplex (the transfer, T-segment) through a transient double-stranded break in another (the gate, G-segment). Here we monitor the passage between short double-stranded DNA segments within long single-stranded DNA circles that leads to catenation of the circles. To facilitate catenation, the circles were brought into close proximity using a tethering oligonucleotide, which was removed after the reaction was complete. We varied the length and the composition of the reacting DNA segments. The minimal DNA duplex length at which we detected catenation was 50–60 bp for DNA gyrase and 40 bp for topoisomerase IV (Topo IV). For Topo IV, catenation was observed when one, but not both, of the DNA-DNA duplexes was replaced by a DNA-RNA duplex. Topo IV cleaved the DNA-DNA duplex, but not the DNA-RNA duplex implying that the DNA-RNA duplex can be a T-segment but not a G-segment.

Received for publication, April 26, 2006 , and in revised form, June 21, 2006.

^* This work was supported by National Institutes of Health Grant R37 CM31657 (to N. R. C.) and a short-term fellowship of Human Frontier Science Program (to P. B. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The online version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.

In memorium.

¹ To whom correspondence should be addressed: CNRS UMR 5153; Muséum National d'Histoire Naturelle USM0503; INSERM UR565; 43 rue Cuvier, 75231 Paris cedex 05, France. Tel.: 33-140793859; Fax: 33-140793705; E-mail: arimondo{at}mnhn.fr.

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