Title: DNA double-strand break repair in blood development and lymphocyte diversification
Funder: Medical Research Council

Abstract: We study DNA double-stranded break (DSB) repair, with a focus on the mechanism and context-specific regulation of the homologous recombination (HR) and nonhomologous end joining (NHEJ) pathways. Both pathways are vital in humans: accurate HR ensures a stable genome is preserved throughout development and in proliferating cell populations, while deletional non-homologous end joining (NHEJ) generates genetic diversity within immune repertoire within differentiating and antigen-activated lymphocytes. Cancer-initiating translocations and structural chromosomal rearrangements arise from unscheduled NHEJ events, so an important question is how an appropriate equilibrium is maintained between accurate HR and mutagenic NHEJ between different tissues and cellular contexts. A primary aim of this programme is to understand the context-specific contributions that distinct DNA double-strand break (DSB) repair systems make during the development, diversification and maintenance of blood cell lineages. In lymphocytes, the joining of interspaced DNA breaks induced during V(D)J recombination and immunoglobulin class-switch recombination (CSR) relies on non-homologous end joining (NHEJ). However, stark unexplained differences exist between the proteins required for each process. The repair of AID-dependent DSBs during CSR for example, is unusual in that it relies less on classical NHEJ (c-NHEJ) proteins, yet entirely on a mechanism involving 53BP1 and its downstream effector complex shieldin. Conversely, efficient V(D)J recombination relies on 53BP1 and is completely lost upon c-NHEJ deficiency, yet does not require the shieldin proteins. These observations indicate 53BP1-dependent functions in CSR and V(D)J recombination are mechanistically distinct, yet our understanding of these processes remains poor. By comprehensively characterising the lymphocyte development and antigen-receptor recombination defects that manifest in mice deleted for these NHEJ proteins, we have provided the first evidence for the existence of multiple independently-operating NHEJ pathways. We predict that these different branches needed to evolve in response to the structurally distinct DNA break intermediates generated during V(D)J recombination and class-switch recombination (CSR). Ultimately, we hope to link the unusual specificities of atypical NHEJ factors to the precise DNA lesions they target, additionally illuminating the DNA lesions these proteins target when they trigger cancer development. Human deficiencies in several of the DSB repair proteins we are investigating also manifest in bone-marrow failure, confirming genome-maintenance roles for these factors in blood progenitors. By investigating the DNA lesions/structures resolved by these factors in developing lymphocytes and HSC compartments, a secondary aim of the programme is to elucidate DNA repair mechanisms that are universally important for blood production.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: BLOOD;CANCER AND NEOPLASMS