Molecular characterization of non-canonical ATM signaling

ATM is the protein kinase that is mutated in the disease ataxia telangiectasia (AT). These patients display immune deficiencies, cancer predisposition, neuronal degeneration and radiosensitivity. The molecular role of ATM is to respond to DNA double-strand breaks (DSBs) and alterations in chromatin structure by phosphorylating its targets, hereby promoting repair of DNA damage or arrest of the cell cycle. ATM is activated by two known co-factors in a stimulus dependent manner: following the induction of DSBs, NBS1 (mutated in Nijmegen breakage syndrome and part of the MRN complex) is required for activation of ATM. ATM can also be activated in the absence of DNA DSBs. Treatment of cultured cells with hypotonic or replicative stress leads to the activation of ATM, presumably because these agents induce changes in chromatin structure, in an NBS1 independent manner. Recently we have identified ATMIN, for (ATM INteracteractor) as being required for ATM function after replicative/hypotonic stress. We have shown that non-canonical ATM signalling via ATMIN is required for ATM function in B cells: during their maturation, in class switch recombination and in the suppression of lymphomagenesis. These data show that ATMIN-mediated ATM activation is of physiological relevance both in somatic recombination and in maintaining genomic stability. In my group, I have unpublished data showing that ATMIN mediated-ATM activation is required for the localisation of the ATM substrate 53BP1 into what we call the 53BP1 containing protection complex (53PC). We hypothesize that ATMIN plays a role in the assembly of the 53PC of under-replicated DNA and that this complex functions to shield chromosomal-fragile sites. We aim to understand the molecular mechanism by which ATMIN functions in this non-canonical ATM signalling pathway. We further hypothesize that ATMIN binds to fragile sites within the genome where it co-localises with 53BP1 hence we aim to identify which regions of the genome ATMIN binds to by chromatin immunoprecipitation (ChIP) and ChIP-sequencing. Additionally, we hypothesize that other novel proteins must be required for ATM activation and 53BP1 localisation after replicative stress. To identify such proteins I have taken a new approach of performing a genome-wide siRNA library screen with the read-out being microscope-based (for activated ATM and 53BP1 localisation via immunofluorescence). We aim to characterise the function of these newly identified factors in non-canonical ATM signaling. This proposal will allow us to understand more about how non-canonical ATM signalling functions. To carry out this proposal I request the positions of one postdoctoral scientist with a background in molecular/cellular biology and 1 PhD student both to work 100% of their time on the proposed projects.

DNA replication is a fundamental process of the cell that ensures accurate duplication of the genetic information and subsequent transfer to daughter cells. Our aim within this project was to better understand this process. Using cellular models we showed that a key kinase (termed ATM), responsible for signaling DNA double-strand breaks, indeed functions in signaling DNA replication stress. In doing so, it requires a co-factor (termed ATMIN) in order to phosphorylate its substrates and localise DNA repair proteins to sites of DNA damage. The outcome is to preserve genome integrity in response to replication stress. Because DNA double-strand breaks are generated within lymphocytes, patients with mutations within ATM suffer from immunodeficiency (amongst other pathologies). To better understand ATM regulation within T cells we generated murine models to determine the contribution of both ATMIN and NBS1 to the functions of ATM within T cells. We revealed that the functions of NBS1 are distinct within T cells, yet the combined loss of both ATMIN and NBS1 led to enhanced DNA damage that drove spontaneous peripheral T cell hyperactivation, proliferation as well as excessive production of proinflammatory cytokines and chemokines, leading to a highly inflammatory environment. In vivo, this led to severe intestinal inflammation, colitis and premature death. This project has overall led to a better understanding of ATM regulation, via its co-factors ATMIN and NBS1, in the maintenance of genome integrity and in the suppression of pathology.