Research Groups
ICGEB Molecular Biology Laboratory
Detached Section
Scuola Normale Superiore SNS
c/- Institute of Clinical Physiology
Via Moruzzi 1, 56124 Pisa, Italy
Lab tel: +39-050-3153091
Office tel: +39-050-3153103
Office fax: +39-050-3153327
Research Interests and Description
Research Interests
Regulation of human DNA replication at the molecular level; human DNA helicases; methodologies for gene therapy
Description of Research
Regulation
of human DNA replication at the molecular level
The
Group studies the composition and structure of the replicating complexes
regulating the function of the human replication origins, exploiting in
particular the lamin B2 one. Collaboration with the IGM-CNR of Pavia showed
that a minimum sequence of 129 bp, containing the area covered by the
replicative complex proteins and the start sites of synthesis, suffices to
confer origin function. Within this sequence we have identified the precise
sites of interaction, along the cell cycle, of canonical members of the
replicative complexes and identified the DNA topoisomerases I and II as new
members. More recently, we demonstrated that the homeotic protein HOXC13, that
displays a specific affinity for the lamin B2 origin, interacts in vivo with it (see figure, left panel), but only at the G1/S border,
immediately prior to the synthesis start. DMS-footprinting of the
immuno-precipitated material (see figure, right panel) showed a protection
within the replicative complex area. ChIP assays on seven other origins showed
the presence of the protein in all of them. Thus, the participation of HOXC13
in the replicative complex may be a generalized phenomenon. In agreement with
this contention, GST-pulldown demonstrated an affinity of HOXC13 for the ORC1
and CDC6 proteins, while fluorescence-based studies showed that the protein
co-localizes with the replication foci at the beginning of S and that HOXC13
interacts with ORC2 and CDC6, only at the G1/S border. The genes for HOXC13,
topo I and topo II are all proto-oncogenes; we have observed that also two
other oncoproteins, cMyc and c-fos, are present on the origin in asynchronous
cells. We are now investigating the cell-cycle dependence of the interactions
of these two oncoproteins with the origin and with the other members of the
replicative complexes, and plan to identify other members of these complexes by
combined in vivo and in vitro proteomic approaches. In vivo we will
utilize a cell line, created in collaboration with the Department of Genetics of
the University of Pavia, that contains 500 copies per cell of the lamin B2
origin. In vitro we set up conditions for assembling on the
origin a protein/DNA complex resembling the pre-replicative complex observed in
vivo. We are also studying the chromatin organization of
the origin area in relation to its functional cycle.
Human
DNA helicases
We have
purified ten human DNA helicases, characterized their properties and identified
the genes for seven of these; in collaboration with the Genome Stability Group
of the ICGEB, we have observed that, of the five RecQ helicases, only RecQ1 and
RecQ4 bind the origin, RecQ4 being loaded in late G1, RecQ1 at the onset of S
and both leaving the origin after synthesis start. The precise sites of binding
and the possible interactions with the other members of the replicative complex
will be studied.
Methodologies
for gene therapy
In
collaboration with the IFC-CNR of Pisa we look in two ways for conditions
favouring gene-targeting. (1) Vectors based on the adeno-associated virus (AAV)
show a high efficiency of homologous integration of the therapeutic sequence,
but their usage is hampered by the difficulty of producing sufficient amounts
of virions. We have developed a methodology for the replication of the AAV
genome in Saccharomyces cerevisiae cells and are progressing towards the
possible production of mature virions in yeast. (2) A construct of the yeast
Rad 52 protein containing the membrane-permeating peptide of HIV1 Tat,
introduced directly into cultured human cells, enhances 50-fold the efficiency
of homologous integration. We are studying the conditions to further enhance
this efficiency with a view to couple this methodology with the inherent
tendency to
homologous integration of AAV-based constructs.
Recent Publications
Falaschi, A. Binding of DNA topoisomerases I and II to replication origins. 2009. In: DNA Topoisomerases, Methods in Molecular Biology 582, D.J. Clarke (ed.), Humana Press, Springer Verlag, Heidelberg, Germany. In press
Comelli, L., Marchetti, L., Arosio, D., Riva, S., Abdurashidova, G., Beltram, F., Falaschi A. 2009. The homeotic protein HOXC13 is a member of human DNA replication complexes. Cell Cycle 8, 454-459
Falaschi, A. 2008. Similia similibus: pairing of homologous chromosomes driven by the psychochemical properties of DNA. HSFP Journal 2, 257-261
Abdurashidova, G., Radulescu, S., Sandoval, O., Zahariev, S., Danailov, M.B., Demidovich, A., Santamaria, L., Biamonti, G., Riva, S., Falaschi, A. 2007. Functional interactions of DNA topoisomerases with a human replication origin. EMBO J. 26, 998-1009
Falaschi, A. 2007. Changing disciplinary borders into frontiers of progress. Editorial. HFSP Journal 1, 1-3
Falaschi, A., Abdurashidova, G., Sandoval, O., Radulescu, S., Biamonti, G., Riva, .S. 2007. Molecular and structural transactions at human DNA replication origins. Cell Cycle 6, 1705-1712
