Oxidative stress and metabolic reconfiguration in the development of diseases with underlying deficiencies in DNA repair.

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Our own previous studies indicate that depletion of NAD+ due to hyperactivation of poly(ADP-ribose) polymerase, as a consequence of unrepaired DNA damage, has a profound effect on the redox status of a cell leading to raised levels of reactive oxygen species (ROS). One of the main and most relevant targets of ROS is DNA, leading to DNA damage which may then be fixed as mutations. Cells unable to deal efficiently with DNA damage, such as those with an underlying deficiency in DNA repair enzymes, seemingly have a double challenge: the primary genotoxic insult and the ensuing oxidative stress. One aim of this proposal is to examine the role of poly(ADP-ribose) polymerase hyperactivation and oxidative stress in diseases which are deficient in key players in the repair of DNA double-strand breaks: ATM, NBN, XLF, LIGIV, FANCD1/BRCA2, FANCN/PALB2 etc. The effect of permanent stress due to DNA damage is often compensated for by so called metabolic reconfiguration. For example, after DNA damage, activated ATM promotes the pentose phosphate pathway via glucose-6-phosphate dehydrogenase leading to increased NADPH production and improved antioxidant capacity. In this project we will examine a collection of cell lines from distinct, but overlapping, disorders with deficiencies in the repair of DNA double-strand breaks for changes in genes regulated by antioxidant response elements (ARE) in the expectation that ROS arising as normal endogenous metabolites, for example during mitochondrial respiration, might be the target of metabolic reconfiguration. Polymorphisms in AREs will also be examined, particularly with respect to clinical variation in the DNA repair deficiency Nijmegen Breakage Syndrome (NBS). For this we have over 40 cell lines from clinically well characterised patients, all with the same homozygous founder mutation. For many of these cell lines, the amount of hypomorphic NBN protein fragment has been determined and correlated with clinical course, particularly cancer incidence.