Repair of oxidative DNA damage is delayed in the Ser326Cys polymorphic variant of the base excision repair protein OGG1

Rachael M. Kershaw 0 Nikolas J. Hodges 0 0 School of Biosciences, University of Birmingham , Vincent Drive, Edgbaston, Birmingham, B15 2TT , UK TThheeAAuutthhoorr22001122..PPuubblilsishheeddbbyyOOxxfofordrdUUninvievresristiytyPrPersesssononbebheahlafloffotfhethUeKUKEnEvnirvoinromnemnetanltaMl uMtaugtaegneSnoScioectiye.ty. AAllll rriigghhttss rreesseerrvveedd.. FFoorr ppeerrmmiissssiioonnss,,pplleeaasseeee--mmaaiill::jjoouurrnnaallss..ppeerrmmiissssiioonnss@@oouupp..ccoomm.. - *To whom correspondence should be addressed. Tel: 0121 414 5906; Fax: 0121 414 5925; Email: Received on September 29, 2011; revised on February 8, 2012; accepted on February 9, 2012 Geneenvironment interactions influence an individuals risk of disease development. A common human 8oxoguanine DNA glycosylase 1 (OGG1) variant, Cys326hOGG1, has been associated with increased cancer risk. Evidence suggests that this is due to reduced repair ability, particularly under oxidising conditions but the underlying mechanism is poorly understood. Oxidising conditions may arise due to internal cellular processes, such as inflammation or external chemical or radiation exposure. To investigate wild-type and variant OGG1 regulation and activity under oxidising conditions, we generated mOgg12/2 null mouse embryonic fibroblasts cells stably expressing Ser326- and Cys326-hOGG1 and measured activity, gene expression, protein expression and localisation following treatment with the glutathione-depleting compound L-buthionine-S-sulfoximine (BSO). Assessment of OGG1 activity using a 7,8-dihydro-8-oxodeoxyguanine (8-oxo dG) containing molecular beacon demonstrated that the activity of both Ser326- and Cys326-hOGG1 was increased following oxidative treatment but with different kinetics. Peak activity of Ser326-hOGG1 occurred 12 h prior to that of Cys326-hOGG1. In both variants, the increased activity was not associated with any gene expression or protein increase or change in protein localisation. These findings suggest that up-regulation of OGG1 activity in response to BSO-induced oxidative stress is via post-transcriptional regulation and provide further evidence for impaired Cys326-hOGG1 repair ability under conditions of oxidative stress. This may have important implications for increased mutation frequency resulting from increased oxidative stress in individuals homozygous for the Cys326 hOGG1 allele. Introduction Reactive oxygen species (ROS) are essential for cellular processes, including gene regulation, cell-mediated immunity, cell differentiation, post-translational processing of proteins and cellular signalling (1,2). ROS are generated via endogenous sources including the electron transport chain, oxidase enzymes and phagocytes as well as through environmental exposure to exogenous sources including ultraviolet (UV) light, ionising radiation, metals and polycyclic aromatic hydrocarbons. ROS can induce a range of mutagenic DNA lesions, including abasic sites, DNA strand breaks and base oxidations (3). Due to its low oxidation potential, guanine is the most easily oxidised base (4) and the two most abundant products formed are 2,6-diamino-4-hydroxy-5-formamidopyrimidine (fapyG) and 7,8-dihydro-8-oxodeoxyguanine (8-oxo dG), with subsequent oxidation reactions resulting in the formation of spiroiminodihydantoin and guanidinohydantoin (5). In normal tissues, it is estimated that the steady-state rate of formation of 8-oxo dG lesions is 103 per cell per day and this can be as high as 105 lesions per cell per day in cancer tissues (6). 8-Oxo dG is mutagenic because it can mispair with adenine during DNA replication and lead to G:C/T:A transversion mutations (7,8). As detailed in a recent review (9), accumulation of oxidative damage has been shown to contribute to the process of normal cellular ageing (10) and various degenerative diseases, including cancer (11), Alzheimers disease (12), Parkinsons disease (13) and cardiovascular disease (14). Mammalian repair of 8-oxo dG occurs via the short patch base excision repair (BER) pathway initiated by 8-oxoguanine DNA glycosylase 1 (OGG1), a bifunctional glycosylase/AP lyase which recognises oxoG:C pairs and catalyses both the removal of 8-oxo dG and the cleavage of the DNA backbone (15). Although capable of bifunctional activity, recent evidence suggests that the AP lyase activity of hOGG1 is not essential and that the enzyme may operate as a monofunctional glycosylase in vivo (16). Subsequent activities by apurinic/ apyridimic endonuclease 1 (APE 1), b-polymerase and DNA ligase I result in repair completion (17). The human OGG1 gene (hOGG1) undergoes alternative splicing to generate two major isoforms: the nuclear a-hOGG1 (hOGG1-1a) and mitochondrial b-hOGG1 (hOGG1-2a) (18 20). The hOGG1 gene has been mapped to chromosome 3p26.2 (21), a region frequently subject to monoallelic deletion and loss of heterozygosity in a number of cancers (22,23). Importantly, reduction in activity has been associated with increased risk of cancer (24,25). The mOgg1 / null (KO) mouse is viable, but compared with wild-type has greater levels of 8-oxo dG, shows increased G:C/T:A transversion mutations in genomic DNA in non-proliferative tissues and is predisposed to lung adenocarcinoma and adenoma (2628). There has therefore been interest in identifying mutations in the hOGG1 gene and investigating their effects on levels and activity of the protein (29). Several polymorphisms of the hOGG1 gene have been identified (30). A single-nucleotide polymorphism at Codon 326 (S326C), present at an allele frequency of 0.330.45 in Asian populations and 0.220.27 in Caucasian populations, occurs due to a C/G substitution at position 1245 in Exon 7 and results in the exchange of a cysteine for a serine in Codon 326. Epidemiological evidence for an association between the S326C allele and cancer susceptibity is conflicting. Under normoxic conditions, individuals homozygous for the S326C hOGG1 allele have been shown to have an increased risk of cancers including oropharangeal, nasopharangeal, oesophageal and lung (3137) but to have no increased risk of breast, biliary tract or colon cancers (3842). Under normoxic conditions, reduced repair ability of S326C OGG1 has been observed by some (43,29) but not others (4451) although catalytic efficiency (Kcat/Km) for excision of 8-oxo dG was found to be 1.6-fold lower for purified Cys326-a-hOGG1 protein (45). A recent analysis of epidemiology data available for lowpenetrance variants in DNA repair genes and cancer susceptibility found inconclusive evidence for an association between S326C and cancer risk (52). However, as well as inherent problems such as study design differences, bias and chance, heterogeneity in epidemiological data could be due to gene gene interactions and geneenvironment interactions which are difficult to control for. Interestingly, in studies where factors including alcohol, meat consumption and smoking status were controlled for, an (...truncated)