All living organisms are constantly exposed to physical and chemical sources that challenge the integrity of the genome. Considering the high number of chemical and physical insults potentially deleterious to which cells are constantly exposed, the maintenance of genome stability is for all living organisms a main challenge during the cellular life cycle. The ability to cope with DNA damage is crucial for cellular proliferation and, in higher eukaryotes, the loss of function of genes responding to DNA damage often results in genetic syndromes and cancer predisposition. Recognition of DNA damaged structures and their accurate repair are two crucial events that involve several factors and multiple specialized pathways. These events are finely orchestrated by the cell cycle checkpoints aimed to sense DNA damage, arrest cellular proliferation and activate the most accurate repair pathway. At DNA double strand breaks (DSB, the most cytotoxic lesions), homology-directed repair initiates with the 5’ strand nucleolytic degradation of the broken end, a process called resection. In all the eukaryotes, resection is tightly regulated and, not surprisingly, mutations in resection machinery genes are associated with high genome instability and therefore cancer predisposition. In the past we proposed that the Rad9 checkpoint factor, through the interaction with modified histones physically inhibits the ssDNA accumulation at DSB. Importantly, this function is conserved with the mammalian counterpart 53BP1. In this thesis, using budding yeast as model system, I have been involved in three projects focusing on the role of Rad9 in DSB repair pathway choice and how the chromatin positioning of this factor is dynamically regulated in response to these lesions. In a first part, I collaborated in the comprehension of Rad9 genetic and functional interactions with different repair factors during DSB metabolism. In brief, we found that Rad9 positioning around DSB ends are important for tethering of DSB ends, resection start and, most importantly, recruitment of recombination factors. Our findings provided a molecular explanation how Rad9 inhibition facilitates Homologous Recombination (HR), preventing the Non Homologous End Joining repair (NHEJ). Later, I studied the role of the Slx4-Rtt107 complex in modulating checkpoint signaling and nucleolytic processing during homology-directed repair of DSBs. Using different genetics and biochemical approaches, I described a novel Slx4 function in supporting DSB resection through the inhibition of the formation of a complex between Rad9 and the checkpoint factor Dpb11 (TOPBP1 in mammals). In mammals, biallelic mutations in SLX4 are associated with the Fanconi Anemia, a genetic disorder associated with defects in DNA repair and high cancer risk. Considering this, our results may be important for understanding how Slx4 protects genome stability and favors cellular proliferation in human beings. In the last part, I have been involved in an international collaboration with Dr. Marcus B. Smolka (Cornell University, Ithaca, NY, USA). Here I studied the role of Dpb11 in coordinating the recruitment of Rad9 during the resection process. We found that a constitutive interaction between Dpb11 and Rad9 severely abrogates ssDNA accumulation in cells responding to DSB lesions, suggesting that this interaction is a crucial point of regulation regarding this process. In human cells, SLX4 shares functional homology with BRCA1, whose interaction with TOPBP1 is mutually exclusive with TOPBP1-53BP1. Our results suggest that TOPBP1, through the coordinated recruitment of pro- and anti-recombination factor, is an essential regulator of DNA repair and genome stability.
DYNAMIC BINDING OF RAD9/53BP1 ON DNA LESIONS PROMOTES ACCURATE REPAIR AND GENOME STABILITY / D. Dibitetto ; supervisor: F. Marini A. Pellicioli. - Milano : Università degli studi di Milano. DIPARTIMENTO DI BIOSCIENZE, 2017 Feb 10. ((29. ciclo, Anno Accademico 2016.
|Titolo:||DYNAMIC BINDING OF RAD9/53BP1 ON DNA LESIONS PROMOTES ACCURATE REPAIR AND GENOME STABILITY|
|Data di pubblicazione:||10-feb-2017|
|Parole Chiave:||DNA damage; Rad9; 53BP1; Homologous Recombination; DNA repair; genome stability|
|Settore Scientifico Disciplinare:||Settore BIO/11 - Biologia Molecolare|
|Citazione:||DYNAMIC BINDING OF RAD9/53BP1 ON DNA LESIONS PROMOTES ACCURATE REPAIR AND GENOME STABILITY / D. Dibitetto ; supervisor: F. Marini A. Pellicioli. - Milano : Università degli studi di Milano. DIPARTIMENTO DI BIOSCIENZE, 2017 Feb 10. ((29. ciclo, Anno Accademico 2016.|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.13130/dibitetto-diego_phd2017-02-10|
|Appare nelle tipologie:||Tesi di dottorato|