Among many oxidized bases, 8-oxoG has been intensively studied as it is implicated in various diseases, such as cancer, neuronal degeneration and teratogenicity. 8-oxoG has mutagenic potential and various enzymes specifically act on the lesion (3
). In the present study, we propose that 2-OH-A, an oxidized form of adenine, must also be involved in biological responses, as 2-OH-A in DNA can be efficiently excised by a DNA glycosylase encoded by the hMYH
2-OH-A, also known as isoguanine, has been detected in DNA from isolated human tissues and from experimental animals and its amount was increased by exposure to various sources of reactive oxygen species, both in vitro
and in vivo
). Furthermore, it has been shown that in certain human cancerous tissues, amounts of 2-OH-A are increased several-fold compared to findings in normal non-cancerous tissues (50
). Concerning the origin of 2-OH-A, the extent of oxidation of the 2 position of the adenine base by hydroxyl radicals is higher in its free nucleotide form than in DNA (47
), thereby indicating that misincorporation of 2-OH-dATP from the nucleotide pool is the major source of 2-OH-A in DNA. 2-OH-A in DNA thus incorporated forms with a relatively stable base pair with thymine and cytosine (51
), and may also pair with the syn
forms of guanine and adenine (47
We reported that 2-OH-dATP is efficiently hydrolyzed to 2-OH-dAMP by hMTH1, originally identified as 8-oxo-dGTPase (31
), indicating that organisms come equipped with an enzyme to eliminate 2-OH-dATP from their nucleotide pools. Since hMTH1 has the lowest Km
value with 2-OH-dATP among its substrates and E.coli
MutT, a prototype of 8-oxo-dGTPase, has little ability to hydrolyze 2-OH-dATP, one could argue that human and mammalian cells are exposed to a higher risk of incorporation of 2-OH-dATP into their genome than are E.coli
cells, thus they eliminate the precursor from the nucleotide pool. Since 2-OH-A in DNA forms a stable Watson–Crick base pair with thymine, it has been speculated that 2-OH-A paired with thymine in DNA may escape repair (51
). However, Jaruga and Dizdaroglu (53
) reported that 2-OH-A detected in DNA of human cells after H2
exposure is repaired within 4 h to background level, indicating that human cells possess repair activity for 2-OH-A in DNA.
In the present work, we examined an enzyme activity that introduces an alkali-labile site into 2-OH-A-containing oligonucleotides and found evidence for such a DNA repair activity that is likely to act as a DNA glycosylase. Furthermore, we showed that the repair activity is co-purified with hMYH and that recombinant hMYH itself has such activity. hMYH lacks AP lyase activity, thus we suspect that one could not detect the repair activity for 2-OH-A without alkaline treatment (54
Our data indicate that hMYH (2-OH-A/adenine DNA glycosylase) recognizes 2-OH-A paired with 8-oxoG or purines as well as adenine paired with guanine or 8-oxoG. hMYH in partially purified fractions preferentially acts on adenine paired with guanine in DNA rather than that paired with 8-oxoG; however, it binds more efficiently to substrates containing the latter. These characteristics of authentic hMYH are the same as those of calf MYH (44
). However, recombinant hMYH in crude extracts exhibited strongest activity for oligonucleotides containing adenine paired with 8-oxoG, as reported by others (19
), but substantially less nicking activity for oligonucleotides containing adenine or 2-OH-A paired with guanine. Thus, the partially purified fraction of hMYH from Jurkat cells may contain some other factors which modify its substrate specificity.
It has been shown that the C-terminal region of E.coli
MutY (amino acids 227–350), the sequence of which is homologous to hMYH (amino acids 352–535 in hMYHα3) and partly to MutT (Fig. ), specifically binds 8-oxoG opposite adenine or an abasic site (55
). hMYH binds strongly to oligonucleotides containing adenine paired with 8-oxoG and to a lesser extent when paired with guanine, but it is likely that hMYH quickly dissociates from those with 2-OH-A paired with guanine after excising 2-OH-A. This may explain why the 2-OH-A-containing oligonucleotides but not the others were degraded by E.coli
nucleases after incubation with the recombinant hMYH extract, as seen in Figure B.
Figure 8 Sequence alignment of the E.coli MutT, hMTH1 and hMYH C-terminal regions (amino acids 341–501) and the E.coli MutY C-terminal region (amino acids 211–350). The alignments for MutT, hMYH and MutY are based on the data of Noll et al. (55) (more ...)
In crude extracts from E.coli
cells, we found no activity on substrates containing 2-OH-A (Fig. B). We then asked whether purified MutY acts on 2-OH-A, but no such activity was detected (T.Ohtsubo, H.Kamiya, H.Kasai and Y.Nakabeppu, unpublished results). Thus, hMYH but not MutY has a novel repair activity for 2-OH-A in DNA, as does hMTH1 for 2-OH-dATP (31
). Interestingly, we found that there are significantly conserved residues between hMYH (amino acids 369–499 in hMYHα3) and the entire hMTH1 molecule, as shown in Figure . Thirty-nine residues in hMTH1 (156 amino acids) are identical to those in the hMYH C-terminal region and 18 of them are also conserved in E.coli
MutT and MutY, suggesting that the 18 residues are involved in binding to 8-oxoG. The 21 residues conserved only between hMYH and hMTH1 may determine the specificities of the two proteins for 2-OH-A and 2-OH-dATP, in addition to 8-oxoG and 8-oxo-dGTP.
In human cells, authentic hMYH was detected in nuclei and mitochondria by western blotting and their molecular weights differed, thereby indicating that there are multiple forms of hMYH. Based on 5′-RACE and RT–PCR of hMYH transcripts, we conclude that there are three major hMYH transcripts, namely hMYH α, β and γ, with a different 5′ sequence or first exon, and further that each transcript is alternatively spliced, thus forming over 10 mature transcripts. A major transcript, hMYHα3, essentially corresponds to the hMYH cDNA originally reported (18
) and encodes two polypeptides, p54 and p53, the former translated from the first and the latter from the second AUG.
It has been reported that the translation product of the hMYHα3 transcript is exclusively localized in mitochondria and that the N-terminal sequence of hMYH translated from the first AUG functions as a mitochondrial targeting signal (19
); however, the authentic hMYH polypeptide detected in mitochondria is p57. The hMYHα1 transcript has a 33 nt insertion into hMYHα3 and thus encodes a polypeptide with an expected molecular mass of 60 031; this may be the p57 detected in mitochondria. Moreover, the nuclear form of hMYH, p52, partially purified from Jurkat cells, is likely to correspond to p53 translated from the second AUG and the hMYH β1, β3 and γ2 transcripts, which are missing the first AUG, may encode the nuclear form of p52 hMYH. Of course, it is also possible that the hMYHα3 transcript produces p53 and p54 by alternative translation initiation, and one of them may correspond to the p52 detected in nuclei of Jurkat cells. Determination of the primary sequences of purified hMYH will reveal which polypeptide is encoded by each hMYH transcript. It is likely that hMYH in mitochondria associates with the inner membrane structure, as does hOGG1-2a (29
), suggesting that the machinery needed for base excision repair in mitochondria generally associates with the inner membrane to achieve efficient repair of mitochondrial DNA.
To date, human cells have been found to be equipped with a repair enzyme, hMYH, for 2-OH-A, as well as hMTH1 for 2-OH-dATP (31
). We will now extend our search for 2-OH-A DNA glycosylase and 2-OH-dATPase activities to other mammalian cells in order to determine whether MYH and MTH1 have the same activities in other mammalian cells.