Role of type II topoisomerases in regulation of supercoiling and

pre-catenation in replication intermediates of DNA

Vctor Martnez1, Jorge Cebrin2, Maridian J. Kadomatsu-Hermosa1, Cristina Parra1, Alicia Castn2, Mara Jos FernndezNestosa1, Christian Schaerer1, Pablo Hernndez2, Dora B. Krimer2 and Jorge B. Schvartzman2
1 Laboratorio de Computacin Cientfica y Aplicada, FP-UNA, San Lorenzo, Paraguay
2 Centro de Investigaciones Biolgicas (CSIC), Ramiro de Maeztu 9, 28040, Madrid, Espaa

ABSTRACT. During replication, DNA molecules undergo Using the result of the computer simulations we obtained the
topological changes that affect supercoiling, catenation and
knotting. To better understand the function of the enzymes that
control the topology of DNA during replication, two-dimensional
agarose gel electrophoresis was used to examine a bacterial
plasmid containing the replication fork stalled after replication of
60 % of the molecule and divides it in the replicated region and
the non-replicated region. The DNA was isolated and treated in
vitro with two type II DNA topoisomerases: topoisomerase IV
(Topo IV) and DNA Gyrase. The effects of these enzymes on the
topology of the replication intermediates and computer
simulations based on the Metropolis Monte Carlo method helped
us to predict the thermodynamic stability of these molecules.

potential energy curve vs. writhe to determine the enthalpy

variation that can be stored in the non-replicated region. We
also found the potential energy curve vs catenation number in
nicked chains, to get the enthalpy variation that can be stored
on the replicated region. Once we obtained the enthalpy curves
for supercoiling and catenanes it was possible to guess which
was the most stable conformation for an replication
intermediate under the effects of both topological phenomena.
This method could be useful to interpret the signals observed in
two-dimensional agarose gels.

Figure 1: pBR322@AatII plasmid with Ter-Tus complex at the recognition site AatII enzyme,
which arrested the replication fork when the 60% of the molecule has been replicated. The
name, mass and genetic map of the plasmid used are indicated on the left side. Inside the map show
the relative position of its most relevant features: the ColE1 unidirectional origin (ColE1 Ori), the E.
coli terminator sequence (TerE) and the ampicillin- and tetracycline-resistance genes (ampR and
tetR). On the right side, an interpretative diagram is shown with the replicated arms arrested at the
60% of the mass.

Figure 4: Metropolis Monte Carlo (MC) simulation. A) Wormlike chain model for simulation of DNA molecules. This model represents the molecule as a succession of rigid
segments, which have bending angles between two consecutive segments. The trial motions used in the wormlike chain model are rigid rotations at an angle about an axis
connecting the points m1 and m2. B) Potential elastic energy E calculation in the MC procedure; kb, T, , C, n and L are constants, is the angle between successive segments in
the worm like model, Lk represents the change in linking number and the writhe Wr has to be determined for each simulation step. The value of enthalpy variation H is the
difference between the elastic energies of supercoiled and relaxed molecules. The free energy G only depends on the Lk value. C) Graphic representation of the simulated
values of Wr / Lk as a function Lk. Data from 100 simulations with 105 trial movements for topoisomers with different values of Lk. D) Profiles of crossing angles as a function
of Lk. Data taken from 100 simulations with 105 trial moves for each simulation for topoisomers with Lk=0 to Lk=20. E) Thermodynamic properties of the unreplicated region
and their relation to the Lk. The elastic potential energy (E) is shown in purple, the enthalpy (H) in red and the free energy (G) in blue.

B
A

Figure 2: Electron Microscopy allows the identification of the

accumulated Replication Intermediate (RI) of pBR322Ter@AatII. On
the left side, visualization of RI containing the replication fork stalled after
replication of 60 percent of the molecule. In the interpretative diagram
shown at the right, the parental duplex is drawn in green and yellow
whereas the nascent strands are drawn in red. Numbers indicate the
relative size of each arm.

Figure 5: Typical conformations of 1.8 kb DNA

molecules with increasing. A) Simple plectonemic forms.
B) Branched plectonemic forms.

Figure 6: Comparison of the enthalpy variation between the regions of the RI A) Both
regions of the RI were simulated separately. For the non-replicated region we performed 100
separate runs with 100,000 trial moves for each topoisomer with Lk values ranging from 0 to 20. For the replicated region, we performed 10 independent runs with 100000 trial moves and Ca
values from 0 to 20 maintaining Lk = 0. B) Profiles of potential energy of the non-replicated (red)
and replicated (black) regions. The abscissa corresponds to the writhe (Wr) and the catenation
number (Ca) of the non-replicated and replicated regions, respectively. The energy curves for
non-replicated and replicated regions were used to determine the most stable conformation for an
RI under the effects of supercoiling and catenation. Note that if the energies stored in the two
regions are equivalent then the number of crossings in the unreplicated region must be equal to
the catenation number of the replicated region.

CONCLUSIONS
Figure 3: Cartoons illustrating the topology sign and handedness of duplex-duplex
intertwining in supercoiled RIs. (A) CCRI from a TopoIV proficient strain in vivo where the sister
duplexes are able to rotate freely around each other at the forks. (B) CCRI from a TopoIV proficient
strain in vitro after deproteinization. In the unreplicated portion, the parental duplex wounds in a
right-handed manner whereas sister duplexes wound in a left-handed manner in the replicated
portion. (C) CCRI from a Topo IV deficient strain in vivo and in vitro after deproteinization. In the
unreplicated portion, the parental duplex wounds in a right-handed manner and the sister duplexes
wound in a right-handed manner too in the replicated portion. Arrows indicate directionality and all
the nodes have a negative sign. Parental duplexes are drawn in blue and green whereas nascent
strands are depicted in red.

The computational simulation using the Metropolis Monte Carlo method demonstrated that the non-replicated
region adopts the plectonemic conformation in thermal equilibrium.
The simulation of the replicated region shows that even in relaxed state (Lk = 0) nascent chains acquire an
induced Wr due to catenation.
The computational simulation of an RI, representing their non-replicated and replicated region separately
confirms that under the condition of thermodynamic equilibrium, the writhe Wr and the catenation number Ca
remain with similar numerical values at all times.