Alessandra Montecucco
Alessandra Montecucco
e-mail:
affiliation: Istituto di Genetica Molecolare-CNR, Pavia
research area(s): Molecular Biology, Cell Biology
Course: Genetics, Molecular and Cellular Biology
University/Istitution: Università di Pavia
Alessandra Montecucco received the degree cum laude in Chemistry and Pharmaceutical Technology from the University of Pavia, Italy in 1984 and the post-graduate specialization program degree in Toxicology from the University of Milano, Italy in 1987. She got the PhD in Genetics and Molecular Biology from the University of Pavia in 1991. She was Researcher at the Istituto di Genetica Biochimica ed Evoluzionistica of the Italian National Research Council (CNR) from 1992 to 2001 and since 2001 she is Senior Researcher at the Institute of Molecular Genetics of CNR in Pavia, Italy. She was EMBO Fellow at the Institut de Recherches Scientifiques sur le Cancer, IRSC-CNRS, Villejuif, France in 1990, Visiting Scientist at the IRSC-CNRS (UPR 42) Villejuif, France in 1994 and Visiting Scientist at the Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms (UK) in 1995.
She has worked for several years on the biochemical and functional characterization of proteins involved in DNA replication and DNA repair in human cells. More recently she has studied the checkpoint pathways that control the sub-nuclear distribution of replication and repair factors in response to DNA damages in human cells and the cross talk between checkpoint pathways and DNA replication stress.
BIOCHEMICAL AND FUNCTIONAL CHARACTERIZATION OF ATP-DEPENDENT DNA LIGASES
DNA ligases catalyze the joining of single-strand or double-strand breaks between adjacent 3’-hydroxyl and 5’-phosphate termini in the phosphodiester backbone of double-strand DNA. For this reason they play a vital role in DNA metabolism. Most eukaryotic DNA ligases use ATP as a cofactor. In mammalian cells there are three DNA ligases called DNA ligase I, III and IV. DNA strand breaks can occur as a result either of the direct action of DNA damaging agents or as reaction intermediates during DNA replication, repair and recombination; therefore the sealing of these breaks by DNA ligase is critical for maintaining genome integrity. Most of the work carried out in the Alessandra Montecucco’s group has led to the characterization of human DNA ligase I that is required for chromosomal DNA replication as well as for several DNA-repair pathways.

MOLECULAR AND CELL BIOLOGY OF DNA REPLICATION
In mammalian cells DNA replication takes place at discrete nuclear sites called replication foci where newly synthesized DNA accumulates. Colocalization on replication foci of replicative enzymes gave rise to the idea that replication takes place within factories. Replication factories assemble in response to demand and their appearance precedes DNA synthesis at specific nuclear sites. The number and size of the replication factories vary throughout S phase according to a program that reflects the replication of various portions of the genome. Our group contributed to the analysis of several aspects. We have identified a short motif, that we called replication factory targeting sequence (RFTS), as the determinant sufficient to target a protein to replication factories. The identification of the RFTS opens the possibility to disassemble the factories by targeting specific peptides. This perspective could be relevant in the search of new anti-proliferative drugs. The RFTS overlaps an evolutionary conserved binding site for PCNA. We have also shown that cell cycle dependent phosphorylation of replicative factors is involved in the dynamic program of replication factories. Finally we have shown that the ordered assembly and disassembly of replication factories is monitored by the cell cycle checkpoints and that the type of DNA damage, its distribution relative to the moving fork and the mechanism involved in the DNA damage recognition could determine the choice between stabilization and dispersal of replication factories in S phase.

CELLULAR RESPONSE TO DNA DAMAGE DURING CELL CYCLE IN HUMAN CELLS
We are currently studying the cell response to chronic replication-dependent DNA damage using as a model system 46BR.1G1 cells established from a patient with a genetic syndrome due to replicative LigI haplo-insufficiency. We have extensively characterized these cells and found that they show a delayed maturation of Okazaki fragments, which results in the accumulation of single- and double-stranded DNA breaks. Notably, the replication-dependent DNA damage in LigI-deficient cells fails to halt cell cycle progression and to induce apoptosis. Actually, 46BR.1G1 cells display only a moderate delay in cell cycle progression and do not activate the S-phase specific ATR/Chk1 checkpoint pathway while the ATM/Chk2 pathway is constitutively activated at basal level. Thus, it is presently unknown which is the strategy used by LigI-deficient cells to cope with this higher basal level of DNA damage. Preliminary results in our laboratory indicate that a number of pre-mRNA processing factors are regulated during the DNA damage response, shifting the alternative splicing pattern of target genes to control cell survival. This finding highlights the existence of a complex regulatory network that links alternative splicing decisions to DNA replication stress and DNA damage.
1: Leva V, Giuliano S, Baroni A, Camerini S, Crescenzi M Lisa A, Biamonti G, Montecucco A “Phosphorylation of SRSF1 is modulated by replication stress” Nucleic Acids Res (2012) Feb; 40(3):1106-1117.
2: S. Giuliano, P. Iadarola, V. Leva, A. Montecucco, S. Camerini, M. Crescenzi, R. Salvini, A. Bardoni “An insight into the proteome of 46BR.1G1 fibroblasts established from a patient affected by DNA ligase I-deficiency syndrome” Electrophoresis 2012, Electophoresis (2012) Jan; 33(2): 307-315.
3: S. Merlo, G. Barillaro, F. Carpignano, V. Leva, A. Montecucco, S. Surdo, L. M S., G. Mazzini “Investigation of cell culturing on high aspect-ratio three-dimentional silicon microstructures” IEEE J Sel Top Quant (2012) 18:1215-1222.
4: D. Pajalunga, E. M.R. Puggioni, A. Mazzola, V. Leva, A. Montecucco, M. Crescenzi “DNA replication is intrinsically hindered in terminally differentiated myotubes” PLoS ONE July 2010: 5 (7) e1159.
5: M.L. Focarelli, S. Soza, L. Mannini, M. Paulis, A. Montecucco and A. Musio “Claspin inhibition leads to fragile site expression” GCC (2009) Sep 16;48(12):1083-1090.
6: R. Vago, V. Leva, G. Biamonti and A. Montecucco “DNA ligase I and Nbs1 proteins associate in a complex and colocalize at replication factories” Cell Cycle S. 7: Soza, V. Leva, R. Vago, G. Ferrari, G. Mazzini, G. Biamonti, and A. Montecucco “DNA ligase I-deficiency leads to replication-dependent DNA damages and impacts cell morphology without blocking cell cycle progression” Mol. Cell. Biol. (2009) 29: 2032-2041 (2009) Aug 15;8(16):2600-7.
8: Rossi R, Pester J M, McDowell M, Soza S, Montecucco A and Lee-Fruman K. K. “Identification of S6K2 as a centrosome-located kinase” FEBS Lett (2007) 581:4058-4064.
9: A. Montecucco and G. Biamonti “Cellular response to etoposide treatment” Cancer Lett (2007) 252:9-18.
10: Rossi R, Lidonnici MR, Soza S, Biamonti G, Montecucco A ‘The dispersal of replication proteins after etoposide treatment requires the cooperation of Nbs1 with the ATR-Chk1 pathway’ Cancer Res. (2006) 66:1675-1683.
11: B. Vitolo, M.R. Lidonnici, C. Montecucco and A. Montecucco “A new monoclonal antibody against DNA ligasi I is a suitable marker of cell proliferation in cultured cell and tissue section samples” Eur J Histochem (2005) 49:349-354
12: G. Camarda, F. Siepi, D. Pajalunga, C. Bernardini, R. Rossi, A. Montecucco, E. Meccia, and M. Crescenzi “A pRb-independent mechanism preserves the postmitotic state in terminally differentiated skeletal muscle cells” J. Cell Biol., (2004) 167:417-423.
13: M.R. Lidonnici, R. Rossi, R. Mendoza-Maldonado, R. Paolinelli, C. Arcangeli, M. Giacca, G. Biamonti and A. Montecucco “Subnuclear distribution of the largest subunit of the human origin recognition complex during the cell cycle” J. Cell Sci. (2004) 117: 5221-5231
14: G. Ferrari, R. Rossi, D. Arosio, A. Vindigni, G. Biamonti and A. Montecuccco “Cell cycle-dependent phosphorylation of human DNA ligase I at CDK sites” J. Biol. Chem. (2003) 278:37761-37767.
15: I. Frouin, A. Montecucco, S.Spadai, and G. Maga “DNA replication: a complex matter” EMBO Rep. (2003) 4: 666-670.
16: I. Frourin, A. Montecucco, G. Biamonti, U. Hubscher, S. Spadari and G. Maga “Cell cycle-dependent dynamic association of cyclin/cdk complexes with human DNA replication proteins” EMBO J. (2002) 21: 1-10.
17: R. Rossi, A. Montecucco, M. Donzelli, M. Denegri, G. Biamonti and A.I. Scovassi “Dephosphorylation of DNA ligase I during the execution step of etoposide-induced apoptosis” Cell Death and Diff (2002) 9: 89-90.
18: A. Montecucco, R. Rossi, G. Ferrari, A.I. Scovassi, E. Prosperi and G. Biamonti “Etoposide induces the dispersal of DNA ligase I from replication factories” Mol. Biol. Cell (2001) 12: 2109-2118.

Project Title:
Links between DNA damage and splicing/RNA processing.