Binding of two DNA molecules by type II topoisomerases for decatenation

Rupesh Kumar,Damian Parry,Jane E. Riley,Valakunja Nagaraja,Andrew D. Bates

doi:10.1093/nar/gks843

Abstract

Topoisomerases (topos) maintain DNA topology and influence DNA transaction processes by catalysing relaxation, supercoiling and decatenation reactions. In the cellular milieu, division of labour between different topos ensures topological homeostasis and control of central processes. In Escherichia coli, DNA gyrase is the principal enzyme that carries out negative supercoiling, while topo IV catalyses decatenation, relaxation and unknotting. DNA gyrase apparently has the daunting task of undertaking both the enzyme functions in mycobacteria, where topo IV is absent. We have shown previously that mycobacterial DNA gyrase is an efficient decatenase. Here, we demonstrate that the strong decatenation property of the enzyme is due to its ability to capture two DNA segments in trans. Topo IV, a strong dedicated decatenase of E. coli, also captures two distinct DNA molecules in a similar manner. In contrast, E. coli DNA gyrase, which is a poor decatenase, does not appear to be able to hold two different DNA molecules in a stable complex. The binding of a second DNA molecule to GyrB/ParE is inhibited by ATP and the non-hydrolysable analogue, AMPPNP, and by the substitution of a prominent positively charged residue in the GyrB N-terminal cavity, suggesting that this binding represents a potential T-segment positioned in the cavity. Thus, after the GyrA/ParC mediated initial DNA capture, GyrB/ParE would bind efficiently to a second DNA in trans to form a T-segment prior to nucleotide binding and closure of the gate during decatenation.

Highlights

DNA topoisomerases facilitate many DNA transaction processes by altering the topology of DNA molecules, and are essential for cell survival [1,2,3,4,5]
We present evidence for the efficient binding of two separate DNA molecules by M. smegmatis gyrase, a property that is shared with E. coli topo IV, but not with the ‘typical’ E. coli gyrase
In the reaction mechanism proposed for DNA gyrase, the carboxy terminal domain (CTD) of GyrA is involved in wrapping the DNA around the holoenzyme, allowing a T-segment from the same molecule to enter the GyrB clamp [53]

Summary

Introduction

DNA topoisomerases (topos) facilitate many DNA transaction processes by altering the topology of DNA molecules, and are essential for cell survival [1,2,3,4,5]. Type IIA topos are the target of a number of classes of cytotoxic compounds, which act to stabilize the transient double-stranded break, leading to DNA breakage and cell death These include anti-tumour agents targeting the human enzymes [8,9] and anti-bacterial compounds, most notably the quinolones and fluoroquinolones [10,11]. Using this double-strand passage mechanism, type IIA topos can relax both positive and negative supercoiling, with a linking number change of ±2 per cycle [6,7,12], and can catalyse decatenation of the catenated intermediates formed during replication [5,13], and can unknot DNA [14].

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Conclusion