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Ennio Prosperi

 

IGM-CNR

c/o Dipartimento di Biologia e Biotecnologie "L. Spallanzani"
Via Ferrata, 9

27100 Pavia


tel: +39 0382 986267
fax: +39 0382 986430
E-mail: prosperiigm.cnr.it



Scientific Interests


DNA damage response: involvement of cell cycle inhibitors in the DNA repair process

An important point in the cell response to DNA damage is the activation of a signaling pathway leading to cell cycle arrest, and to the activation of DNA repair systems. The CDK inhibitor p21CDKN1A plays an important role in the cell cycle arrest, in a p53-dependent manner. A putative role for p21 into DNA repair process has been suggested, thanks to its ability to interact with Proliferating cell nuclear antigen (PCNA). We are investigating how this interaction is established into cells, and whether this is relevant to DNA processes, like nucleotide excision repair (NER), and base excision repair (BER). We have found that p21-null human fibroblasts are deficient in NER, and are more sensitive to UV-C irradiation than their parental cells. Our recent results show that p21 is recruited to DNA damage sites with kinetics very similar to that of PCNA, and that p21 localization does not affect recruitment of NER factors, like DNA polymerase delta, XPG, or CAF-1. We are now investigating whether p21 may be required to modulate interaction of PCNA with its partners during NER. A similar role for p21 in BER has been envisaged. We have shown (collaboration with A.I. Scovassi) that p21 interacts with PARP-1, and that these proteins compete for PCNA binding: we are investigating how this behavior is relevant to BER.




Regulation of multiple interactions of PCNA at DNA replication sites

PCNA is a protein central to DNA metabolism, because it functions as a platform to keep tethered to DNA most replication and repair factors. A key question is how PCNA may regulate partner binding at the replication fork, as well as during DNA repair. We have induced the expression of p21 fused to GFP in HeLa cells and found that p21-GFP arrest cells at the very beginning of S phase, without interfering with loading of PCNA at replication sites, while binding of the polymerase is inhibited. We have analyzed the composition of multiprotein complex containing PCNA recruited to DNA replication sites during S phase in chromatin (the chromatin-bound fraction). We have found that chromatin-bound PCNA is organized as distinct pools, in which a particular partner (e.g. pol delta, or DNA ligase I) binds to a PCNA trimer in a mutually exclusive manner. We have suggested that partner exchange may occur by a CDK2-mediated hand-off mechanism. We are now investigating the behavior of PCNA in various DNA transactions through the use of fluorescent proteins (PCNA fused to GFP or RFP).




Chemical inhibitors and modulators of cell cycle

A variety of synthetic and natural compounds are today investigated as possible regulators of cell proliferation. We are interested in explaining the molecular mechanisms by which antioxidant substances (e.g. resveratrol), or chemical inhibitors of CDK (e.g. roscovitine), are able to affect cell cycle progression by activation of cell cycle checkpoints.


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