download report - Istituto Pasteur

P a r t i c i p a n t s :
Francesco Chiani, Francesca Di Felice, post-doc fellows;
Elisa Cesarini, Anna D’Alfonso, Francesca Romana
Mariotti, PhD students.
C o l l a b o r a t i o n s :
Department of Biological Chemistry, University of California,
Irvine, USA (Dr. Masayasu Nomura); Department of Biological
Chemistry, University of California, Los Angeles, USA (Dr. Michael
Grunstein).
Report of activity
By fine-tuning the level of DNA supercoiling, DNA
topoisomerases are enzymes involved in DNA replication,
transcription, recombination and chromatin
remodeling. They represent the molecular machines
that manage the topological state of the DNA in the cell.
The interest in DNA topoisomerases derives not
only from their crucial role in managing the DNA
topology, but also from a wide variety of topoisomerase-targeted
drugs that have been identified and
utilized as antimicrobials and anticancer drugs, some
of which are currently in widespread clinical use.
In spite of a low number of reported proteins, we
may still expect that other proteins interacting with
DNA topoisomerase I (Topo I) remain yet unknown.
Our aim is to identify the main functional and/or
physical partners of Topo I by studying the processes
in which Topo I is involved like transcription,
recombination and transcriptional silencing.
Altogether the data will contribute to better clarify
the Topo I activity inside the cell and its possible
interaction with DNA and/or any other partner.
Nucleosomes as physical barriers for cleavage
activity of Topo I in vivo
To further characterize the Topo I activity in vivo,
we wanted to evaluate the possible interference of
Principal investigator: Giorgio Camilloni
Professor of Molecular Biology
Dipartimento di Genetica e Biologia Molecolare
Tel: (+39) 06 49912808; Fax: (+39) 06 49912500
giorgio.camilloni@uniroma1.it
37
Molecular genetics of eukaryotes - AREA 3
DNA topoisomerases as global controller of DNA transactions.
Study of DNA topoisomerase IB and its functional partners
nucleosomal structures on its capability to react with
the physiological state of DNA: chromatin.
It is generally accepted that all sequences in DNA
can be substrates for Topo I relaxing activity, even
though Topo I seems to have a higher reactivity
towards some particular sequences showing structural
features like DNA bending. In order to verify
whether nucleosomes affect Topo I site-specific
cleavages, we studied a natural DNA sequence
repeating 35 times the TTA trinucleotide. Such a
sequence provides: the locally bent TA step, presumably
a good substrate for local Topo I cleavage induction
and an intrinsic flexibility potentially useful to
efficiently assemble nucleosome.
The TTA trinucleotide repeat has proved to be efficiently
cleaved in vitro by Topo I in the repeated tract
and also in its surrounding regions, confirming the
preference of the enzyme for this sequence.
Conversely, an in vivo approach showed a reduced
reactivity of the same sequence towards Topo I.
Thus, it is conceivable to hypothesize that chromatin
structure affects Topo I cleavages. Recently it has
been reported that DNA topoisomerase II is more
efficient than Topo I in releasing topological stress
from nucleosomal substrates; this supports the
hypothesis that nucleosomes may represent a barrier
for Topo I activity.
At this purpose a distinction between the global
relaxing activity and the local site specific cleavage
reaction, should be taken under consideration. In fact
a different chromatin organization in a given substrate
could be not determinant in the whole relaxing
activity of Topo I because the enzyme can
release torsional stress acting on different sites in the
substrate. Conversely, when a given sequence is analyzed
in terms of cleavage activity exerted by Topo
I, the absence/presence of a nucleosome could be
very relevant and this latter point was investigated
by our experimental system.
In order to verify this hypothesis, we studied the in
vivo chromatin organization of the (TTA)35 tract. A