A 55-kDa protein isolated from human cells shows DNA glycosylase activity toward 3, N ⁴ -ethenocytosine and the G/T mismatch

Abstract

Etheno adducts in DNA arise from multiple endogenous and exogenous sources. Of these adducts we have reported that, 1,N 6 -ethenoadenine (A) and 3,N 4 -ethenocytosine (C) are removed from DNA by two separate DNA glycosylases. We later confirmed these results by using a gene knockout mouse lacking alkylpurine-DNA-N-glycosylase, which excises A. The present work is directed toward identifying and purifying the human glycosylase activity releasing C. HeLa cells were subjected to multiple steps of column chromatography, including two C-DNA affinity columns, which resulted in >1,000-fold purification. Isolation and renaturation of the protein from SDS͞polyacrylamide gel showed that the C activity resides in a 55-kDa polypeptide. This apparent molecular mass is approximately the same as reported for the human G͞T mismatch thymine-DNA glycosylase. This latter activity copurified to the final column step and was present in the isolated protein band having C-DNA glycosylase activity. In addition, oligonucleotides containing C⅐G or G͞T(U), could compete for C protein binding, further indicating that the C-DNA glycosylase is specific for both types of substrates in recognition. The same substrate specificity for C also was observed in a recombinant G͞T mismatch DNA glycosylase from the thermophilic bacterium, Methanobacterium thermoautotrophicum THF.The four etheno adducts of DNA and RNA have been of considerable interest to organic chemists and physical scientists due to their physical, chemical and spectroscopic properties which had broad applications in protein-nucleic acid interactions and DNA structure [reviewed by Leonard (1, 2) and refs therein]. These adducts became of major interest when they were found to be formed by a variety of environmental agents, as well as produced endogenously (3-7). Mutagenesis studies have shown a wide range of mutagenic frequency depending on the type of the adduct, type of mutation and the system used for detection and quantitation (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19).For more than one decade, this laboratory has focused on studies on the repair (20-26) and replication͞transcription (8-12) of etheno derivatives of dA, dC, and dG. The differing structures of the etheno adducts and their effect on base pairing and base stacking (27-31) influences both repair and replication. Much of the data has been obtained by using prokaryotic systems, which are not always identical to those now found in the more widely used mammalian systems. It was established earlier in repair studies, by using a human system, that all four etheno adducts were released by HeLa cell-free extracts, indicating that they are substrates for DNA glycosylases (24). After partial purification from HeLa cells, 3,N 4 -ethenocytosine (C) repair activity was found to be separate from 1,N 6 -ethenoadenine (A) repair activity (25), which is a function of alkylpurine-DNA-N-glycosylase (APNG) (22). A knockout mouse lacking APNG was then used as a genetic approach to verify these in vitro data (26). Under these c...

Repair of exocyclic DNA adducts: rings of complexity

“…(36,48,72,76) Overlap between glycosylases is also common as examplified by the in vitro identification of the eC-activity in three human DNA glycosylase, TDG, SMUG1 and MBD4. (52,(54)(55)(56) Although the biological function of these activities remains to be seen, such redundancy of repair is expected to be useful for backing up in vivo.…”

“…(51) Through an extensive purification, the eC-DNA glycosylase was identified as a 55 kDa polypeptide by SDS-PAGE, (52) which is the exact molecular mass of the previously purified human mismatchspecific T(U) Á G-DNA glycosylase, termed thymine-DNA glycosylase (TDG). (53) Moreover, the T Á G and U Á G mismatch glycosylase activities co-eluted with the eC activity in the same fractions, and competition studies suggested that they all reside in the same protein.…”

“…(53) Moreover, the T Á G and U Á G mismatch glycosylase activities co-eluted with the eC activity in the same fractions, and competition studies suggested that they all reside in the same protein. (52) It was then proposed that eC is a substrate for hTDG. (52) This was supported by the finding that the functional homolog of hTDG in Methanobacterium thermoautotrophicum, a thermostable mismatch glycosylase (Mig), also excises eC.…”

“…(52) It was then proposed that eC is a substrate for hTDG. (52) This was supported by the finding that the functional homolog of hTDG in Methanobacterium thermoautotrophicum, a thermostable mismatch glycosylase (Mig), also excises eC. (52) In a separate study, Saparbaev and Laval (54) purified an eC activity to homogeneity from E. coli, and identified it as the previously known Mug protein (also termed as double-stranded uracil-DNA glycosylase, dsUDG), a hTDG homolog.…”

“…hTDG also excises eC paired with G with greater efficiency than T from T Á G, but less than U from U Á G. (52,54) Both proteins can remove eC opposite each of the four bases but with varying efficiencies, with eC Á G the preferred substrate. (52,54,57) Recently, Kavli et al (55) described excision of eC by the human single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), which is only found in higher eukaryotes. In another study, the human methyl-CpG binding domain protein (MBD4 or MED1) also shows a weak activity toward eC but only when the opposite base is G. (56) The biochemical details of these two activities have not been reported.…”