Obiettivo Nucleotide excision repair (NER) is involved in preserving genomic integrity by removing a number of diverse lesions, including those formed by UV-light, various environmental mutagens and certain anti-tumour agents from DNA. NER fulfils an important role in protecting humans from cancer. NER is a complex process that involves the concerted action of over 30 polypeptides and involves the following steps: damage recognition, opening of the DNA around the lesion, dual incision of the damaged strand, repair synthesis and ligation. We will pursue an interdisciplinary project to study mammalian NER in vitro and in vivo. We will design and synthesize photoreactive and fluorescent NER substrates and investigate how they interact with NER factors. The novelty of this project involves design of NER substrates containing bulky photoreactive damages and their application to study mammalian NER system. Photocrosslinking studies using photoreactive probes as damages and in proximity to the damage will provide a picture of how NER factors (XPC-HR23B,TFIIH, XPA, RPA, DDB1/2 and others) and their subunits are connected with NER damages or to the undamaged strand in the process of damage verification and processing. Using RPA mutants will allow us to determine subunits and domains of RPA critical for functioning of NER machinery. The advantage of this approach consists in the possibility to identify unstable (transient) NER complexes and to define their architecture and hand-off of damage DNA from one protein to another in NER processing. Novel fluorescent, photoreactive or both NER substrates will be used to determine the intermolecular distances between the individual proteins and damaged sites using fluorescence resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy (FCCS) in live cells. FRET and FCCS studies will be performed by introducing fluorescent NER substrates into cells that already express GFP-tagged NER factors. The kinetics of NER factor assembly and their possible dynamic exchange on locally inflicted lesions will be determined by ultra-fast time-lapse imaging of GFP-tagged factors. While in vitro data should provide a detailed picture of damage recognition, complex assembly and incision during NER, in vivo studies will provide information on the dynamics of the assembly process in the cell. We expect that our studies will provide more insight into the NER mechanism and will contribute to close the gap between in vitro and in vivo studies. Our studies will provide more detailed insight into the assembly and architecture of the NER machinery at various stages of the reaction and in more general terms, provide knowledge into how an intricate protein complex assembles and performs its enzymatic activity in mammalian cells. The data resulted from this project will have a high impact to counteract consequences related to human diseases linked to the NER system and may help to design drugs to combat cancer and aging. Parole chiave Biochemistry Enzyme Function Nucleic Acids Proteins Programma(i) IC-INTAS - International Association for the promotion of cooperation with scientists from the independent states of the former Soviet Union (INTAS), 1993- Argomento(i) Data not available Invito a presentare proposte Data not available Meccanismo di finanziamento Data not available Coordinatore ERASMUS UNIVERSITY OF ROTTERDAM Contributo UE Nessun dato Indirizzo DR. MOLENWATERPLEIN, 50 ROTTERDAM Paesi Bassi Mostra sulla mappa Costo totale Nessun dato Partecipanti (2) Classifica in ordine alfabetico Classifica per Contributo UE Espandi tutto Riduci tutto INSTITUTE OF CHEMICAL BIOLOGY AND FUNDAMENTAL MEDICINE Russia Contributo UE Nessun dato Indirizzo PROSPEKT AKAD. LAVRENTIEVA, 8 NOVOSIBIRSK Mostra sulla mappa Costo totale Nessun dato NATIONAL UNIVERSITY OF IRELAND, GALWAY Irlanda Contributo UE Nessun dato Indirizzo UNIVERSITY ROAD GALWAY Mostra sulla mappa Costo totale Nessun dato
