Identification of DNA glycosylase/AP lyase activity of human hNEIL1 that excises 5-formyluracil and 5-hydroxymethyluracil from DNA

Full text: Exposure of DNA to ionizing radiation and chemical oxidants leads to the formation of several thymine hydroperoxides. 5-Hydroperoxymethyluracil formed spontaneously decompose to produce 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5- hmU). Recent studies revealed that 5-foU is potentially mutagenic in bacteria and mammalian cells. Despite its identification as a major radiation product, the biological effects of 5-hmU remain uncertain. Recently, we found that the E. coli Nth, Nei and MutM form Schiff base intermediates with duplex oligonucleotides containing 5-foU or 5-hmU and cleave the strand at the lesion site. Functional homologs of E. coli DNA glycosylases that act on oxidized bases have been identified in human cells. hOgg1, hMPG and hNTH1 show overlapping substrate specificities with E. coli MutM, AlkA and Nth, respectively. Furthermore, recent works revealed the presence of three human orthologs of E. coli Nei, hNEIL1, NEIL2 and NEIL3. Human hNEIL1 recognizes similar substrates as E .coli Nei, which excises formamidopyrimidines from DNA and oxidized pyrimidines such as thymine glycol from oligonucleotides. Our previous works revealed that human cells possess at least two kinds of DNA glycosylase activity that remove 5-foU from DNA and showed that hNTH1 had a DNA glycosylase/AP lyase activity for 5-foU. Human cells should possess another type of 5-foU DNA glycosylase in addition to hNTH1. In this report we show that hNEIL1 has a 5-foU- and 5-hmU-DNA glycosylase activity that excises these lesions from duplex oligonucleotides. The purified hNEIL1 cleaved the 5- foU- and 5-hmU-containing oligonucleotides via b- and d-elimination reactions. The specific activities for 5-foU and 5-hmU were comparable with that for thymine glycol. In addition, the expression of hNEIL1 cDNA could reduce the frequency of spontaneous mutation and the sensitivity to hydrogen peroxide in E. coli nthnei mutant strain