The oxidative DNA lesion 8,5 '-(S)-cyclo-2 '-deoxyadenosine is repaired bythe nucleotide excision repair pathway and blocks gene expression in mammalian cells

Xeroderma pigmentosum (XP) patients with inherited defects in nucleotide ex cision repair (NER) are unable to excise from their DNA bulky photoproducts induced by UV radiation and therefore develop accelerated actinic damage, including cancer, on sun-exposed tissue. Some XP patients also develop a ch aracteristic neurodegeneration believed to result from their inability to r epair neuronal DNA damaged by endogenous metabolites since the harmful UV r adiation in sunlight does not reach neurons. Free radicals, which are abund ant in neurons, induce DNA lesions that, if unrepaired, might cause the XP neurodegeneration. Searching for such a lesion, we developed a synthesis fo r 8,5'-(S)-cyclo-2'-deoxyadenosine (cyclo-dA), a free radical-induced bulky lesion, and incorporated it into DNA to test its repair in mammalian cell extracts and living cells. Using extracts of normal and mutant Chinese hams ter ovary (CHO) cells to test for NER and adult rat brain extracts to test for base excision repair, we found that cyclo-dA is re paired by NER and no t by base excision repair. We measured host cell reactivation, which reflec ts a cell's capacity for NER, by transfecting CHO and XP cells with DNA con structs containing a single cyclo-dA or a cyclobutane thymine dimer at a sp ecific site on the transcribed strand of a luciferase reporter gene. We fou nd that, like the cyclobutane thymine dimer, cyclo-dA is a strong block to gene expression in CHO and human cells. Cyclo-dA was repaired extremely poo rly in NER-deficient CHO cells and in cells from patients in XP complementa tion group A with neurodegeneration. Based on these findings, we propose th at cyclo-dA is a candidate for an endogenous DNA lesion that might contribu te to neurodegeneration in XP.