CCAR2-mediated regulation of CtIP activity on DNA repair

Descrizione del progetto

Decisioni di riparazione del DNA

Per mantenere l’integrità genomica, le cellule hanno sviluppato meccanismi che consentono di riparare i danni al DNA, e in particolare le rotture a doppio filamento, che sono le più pericolose. Esistono due vie coinvolte nella riparazione del DNA: la giunzione delle estremità non omologhe e la ricombinazione omologa. Mentre la prima lega direttamente le estremità spezzate del DNA, la seconda utilizza una sequenza di DNA omologa come modello per riparare le rotture a doppio filamento. Sostenuto dal programma di azioni Marie Skłodowska-Curie, il progetto CC-DNArep si propone di comprendere il modo in cui le cellule decidono quale percorso utilizzare esplorando il ruolo svolto dalla proteina CCAR2 nelle decisioni di riparazione del DNA e studiando la sua interazione con altre molecole. I risultati del progetto forniranno importanti informazioni sulla via di riparazione del DNA, con il potenziale di esercitare implicazioni per la terapia antitumorale.

Obiettivo

Ensuring cellular integrity and genome stability requires tight control of DNA damage repair. Defects in the processes involved lead to the accumulation of mutations that might cause various diseases, including cancer. DNA double-strand break (DSB) is the most severe type of DNA damage, and two main pathways exist to repair it. The non-homologous end joining (NHEJ) is an error-prone repair pathway with little or no DNA processing, which results in mutations of DNA sequences at the repair site. Alternatively, Homologous Recombination (HR) ensures an error-free repair by using the information present in homolog sequences. Thus, maintaining the HR:NHEJ balance and precise regulation of these pathways is essential for cell fitness. Upon DSB, CtIP is recruited to the damaged site and promotes DNA end resection, required to initiate HR. Recently, CCAR2 was identified as an antagonist interactor of CtIP, therefore promoting NHEJ. CCAR2 is a protein without defined catalytic activity but with several functional domains known to interact with other protein partners. However, how CCAR2 binding regulates CtIP activity on DNA damage repair remains elusive. In this project, I propose to study this interaction by a multidisciplinary approach. Biochemical and biophysical single-molecule fluorescent studies will be applied to understand the regulatory function of CCAR2 upon interaction with CtIP. These will be combined with molecular dynamics simulations to obtain further insights into their direct interaction and how different ligands affect the stability of the CCAR2-CtIP complex. Moreover, in vitro findings will be complemented with experiments in human cell lines upon induced DNA damage. Altogether, this project will provide a full mechanistic understanding of CCAR2-mediated regulation of CtIP activity in the cell. This will shed light on essential information regarding how the HR:NHEJ balance can be modulated, which could have an impact on the treatment of several diseases.