The BRCA1 tumour suppressor is understood to function in the error-free repair of DNA lesions by homologous recombination (HR), yet its actual role in this process is ill-defined. Based on recent experimental observations, I propose what may be the critical role for BRCA1 in maintaining genome stability: antagonising the protein 53BP1 during DNA double-strand break (DSB) repair. Upon BRCA1-deficiency, I predict that DSBs normally repaired by HR during the replicative cell cycle phases, are inapp ropriately repaired by 53BP1-dependent error-prone non-homologous end-joining (NHEJ). Such aberrant repair likely produces the chromosomal rearrangements associated with malignant transformation in BRCA1-deficient cells. My proposed experiments have three core aims: 1. Define the antagonistic role of BRCA1 in inhibiting the repair of DSBs by 53BP1-dependent NHEJ. 2. Establish the molecular determinants of 53BP1-dependent NHEJ. 3. Elucidate the involvement of Rif1 in the antagonism of 53BP1 -dependent DSB repair by BRCA1. I anticipate that the proposed study will provide insights into the elusive functions of BRCA1 in maintaining genomic integrity, which will enhance our knowledge of its molecular role as a tumour suppressor. This work might also identify new avenues to exploit in the clinic to treat BRCA1-associated hereditary and sporadic cancers potentially associated with 53BP1-misregulation.

The development of the shear flow assay is crucial for understanding integrin function as it reproduces the forces that the immune cells are subjected to from blood flow. We will use the assay to evaluate T lymphocyte rolling, adhesion and migration on recombinant ligands and then on endothelial cells to mimic the vasculature in vivo. The first goal is to automate the analysis of the cell tracking images, a task that is presently performed manually which is laborious and with potential for inacc uracy. Automation has not been reported in other laboratories using the assay. Concurrently we will use this assay, in comparison with the well-used static adhesion assay, to assess several adhesion molecules implicated in LFA-1 activation. Of particular interest is kindlin-3 that is mutated in LAD-III, an integrin dysfunction syndrome. We will focus on kindlin-3-modified T cells that unexpectedly migrate in the static assay, but have not been tested under shear conditions. The investigation w ill include the use of: antibodies specific to different forms of LFA-1; peptides that disrupt the interaction of different LFA-1 conformations with the cytoskeleton; siRNAs to interfere with expression of adhesion-related proteins; and GFP-tagged constructs tagged to identify cells containing introduced DNA.

Sen1 is a yeast RNA:DNA helicase that acts as transcription termination factor. Additionally, its helicase-domain functions to resolve co-transcriptionally formed RNA:DNA-hybrids and to prevent transcription associated recombination. As Sen1 is sensitive to DNA damaging agents and interacts with the DNA repair protein Rad2, it is possible that Sen1 either acts to coordinate DNA repair with transcription or that it has transcription-independent functions. This project aims at elucidating the effe ct of Sen1 mutation on recombination and DNA damage, how this is related to a pronounced G1 cell-cycle arrest and to its function as transcription termination factor. The human orthologue of Sen1, Senataxin is mutated in patients of two neurological disorders, the eye movement- and coordination-disorder Ataxia Oculomotor Apraxia type II (AOAII), and Juvenile Amyotrophic Lateral Sclerosis (ALS4). Expanding knowledge on Sen1 function in yeast and later Senataxin itself will help to better understa nd the molecular mechanism underlying both clinical phenotypes. First, I plan to delineate the molecular mechanisms through which Sen1 affects genome stability in yeast. Subsequently, I will extrapolate my findings to human and/or mouse cell-cultures with the aim of understanding if the consequences of Senataxin mutation might be explained by cell type-specific preferences for DNA damage repair.