Molecular Characterisation of Single-Strand Break Repair and Related Responses and their Role in Neuroprotection

DNA breakage can lead to gene damage, cancer, and cell death, if not repaired rapidly and accurately. The commonest type of damage arising in cells is the single-strand break; a breakage of one of the two strands that comprise the DNA double helix. Recently, we have identified a direct link between an individual?s ability to repair single-strand breaks and hereditary neurodegenerative disease. These diseases are termed spinocerebellar ataxia with axonal neuropathy-1 (SCAN1) and ataxia oculomotor apraxia-1 (AOA1) and harbour mutations in the DNA repair genes Tdp1 and APTX, respectively. SCAN1 and AOA1 are associated with the progressive degeneration of specific parts of the brain (particularly the cerebellum), resulting ultimately in an inability of affected individuals to walk properly or to control normal movement. In this programme of work we will advance and extend our understanding of the single-strand break repair process, and address directly the relationship between this process and neurological function. This work will shed light on the link between DNA damage and neurodegeneration, and will hopefully provoke novel approaches for the treatment of certain types of neurological disease.

Single-strand breaks (SSBs) arise from a variety of sources including oxidative stress and are the commonest DNA lesions arising in cells (tens of thousands per cell per day). If not repaired, SSBs can block transcription and DNA replication and lead to genetic instability and cell death. Strikingly, recent work from my laboratory has identified that two proteins associated with hereditary neurodegenerative disease are intimate components of the single-strand break repair (SSBR) machinery, and that cells from one of these diseases possess a major defect in chromosomal SSBR. We have thus recently proposed that SSBR is critical for genetic integrity and survival in neurons, and that this process is vital for normal neurological function. These observations also raise the intriguing possibility that SSBR capacity is an aetiological factor not only for pathological neurological conditions but also for normal human ageing. In the current proposal, we will identify and characterise novel polypeptide components of SSBR and related processes to advance and extend our understanding of this critical process. To achieve this we will employ a combination of genetic, biochemical/proteomic, and cellular approaches, and also implement two new techniques that we are developing. The latter will allow us to characterise, for the first time, SSBR at a site-specific chromosomal SSB (e.g. using ChIP analyses) and to dissect this process within a context of defined chromatin structure. In addition, we will test directly and unambiguously our hypothesis that SSBR is critical for normal neurological function. To achieve this, we will examine the importance of SSBR for genetic integrity and cell survival in primary neurons and for normal neurological function in vivo, using mouse model systems.