2-Hydroxyadenine in DNA is a Very Poor Substrate of the Escherichia coli MutY Protein

HIROYUKI KAMIYA 0 1 HIROSHI KASAI 1 0 Graduate School of Pharmaceutical Sciences, Hokkaido University , Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan (Received, July 3, 2000) (Revision received, September 8, 2000) (Accepted, September 11, 2000) 1 Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health , 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan 2-Hydroxyadenine/MutY/MutM/DNA repair/Oxidative damage To test the possibility that the Escherichia coli MutY or MutM protein acts as a 2-hydroxyadenine (2OH-Ade) glycosylase, we treated double-stranded oligodeoxyribonucleotides containing 2-OH-Ade with the E. coli MutY or MutM protein in vitro. We found that a strand with 2-OH-Ade was a very poor substrate of MutY, irrespective of the base in the complementary strand. Moreover, a strand containing adenine or guanine opposite 2-OH-Ade was also rarely cleaved by MutY. The cleavage of oligonucleotides with 2-OH-Ade by MutM was not observed. These results indicate that neither MutY nor MutM plays an important role in the removal of 2-OH-Ade from DNA. - induces GC TA transversions, which are frequently found in reactive oxygen species-induced mutations, through the formation of 2-OH-AdeG pairs1618). Moreover, we recently found that the human MTH1 protein hydrolyzes 2-OH-dATP more efficiently than 8-OH-dGTP19). Thus, the repair of 2-OH-Ade in DNA as well as the removal of 2-OH-dATP from the nucleotide pool are of great interest. The facts that 2-OH-dATP induces GC TA transversions in E. coli16) and that the mutY and mutM genes are mutator genes of a GC TA transversion10,11) suggest the possibility that MutY and/or MutM prevent mutagenesis by 2-OH-dATP by acting as a 2-OH-Ade glycosylase. In this study we treated double-stranded oligodeoxyribonucleotides containing 2-OH-Ade with the E. coli MutY and MutM proteins in vitro to test this possibility. We found that a strand with 2-OH-Ade was a very poor substrate of MutY, irrespective of the base in the complementary strand. Moreover, a strand containing adenine or guanine opposite 2-OH-Ade was also rarely cleaved by MutY. In addition, the cleavage of the oligonucleotides by MutM was not detected. These results indicate that neither MutY nor MutM acts as a 2-OH-Ade DNA glycosylase. MATERIALS AND METHODS Materials T4 polynucleotide kinase was obtained from Toyobo. The MutY and MutM proteins were purchased from Trevigen Inc. Oligonucleotides with 2-OH-Ade and 8-OH-Gua were synthesized as described20,21). All of the oligonucleotides were purified by reverse-phase and anionexchange HPLCs, as described previously20). The sequences of the oligonucleotides are upper strand 5-GGTGGCCTGXCGCATTCCCCAA-3, lower strand 3-CCACCGGACYGCGTAAGGGGTT-5, where XY are the targeted base pairs. Enzyme reactions 5-End labeling was carried out as described20). The MutY reaction mixture contained 20 mM Tris-HCl (pH 7.6), 80 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 3% glycerol, 100 nM double-stranded oligonucleotides, and MutY (one unit is defined as the amount of enzyme required to cleave oligonucleotides containing an AG mismatch at the rate of 100 fmol for one hr at 37C). The MutM reaction mixture contained 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, 100 nM double-stranded oligonucleotides, and MutM (one unit is defined as the amount of enzyme required to cleave oligonucleotides containing an 8-OH-GuaC pair at a rate of 100 fmol for one hr at 37C). Reactions were stopped by the addition of a termination solution (95% formamide, 0.1% bromophenol blue, and 0.1% xylene cyanol). Samples were heated at 95C for 3 min, and then applied to a 7 M urea, 20% polyacrylamide gel. Autoradiograms were obtained with a Fujix BAS 2000 Bio Image Analyzer. RESULTS AND DISCUSSION Oligodeoxyribonucleotides containing base pairs involving 2-OH-Ade were incubated with MutY. As controls, oligonucleotides containing AT, AG, and A8-OH-Gua pairs were also treated. Figure 1 shows the results of a polyacrylamide gel electrophoresis analysis of the MutY reactions. The MutY glycosylase cleaved DNA fragments containing AG and A8-OH-Gua efficiently (lanes 2 and 3). On the other hand, the excision activity for fragments with 2-OH-Ade Fig. 1. Nicking activity of MutY for 2-OH-Ade-containing DNA fragments. Lane 1, DNA size markers (9-11mers); lane 2, AG; lane 3, A8-OH-Gua; lane 4, 2-OH-AdeA; lane 5, 2-OH-AdeG; lane 6, 2-OH-AdeC; lane 7, 2OH-AdeT. AOH and GOH represent 2-OH-Ade and 8-OH-Gua, respectively. Asterisks indicate the base in the labeled strand. was rarely observed. A faint cleaved band was visible for the 2-OH-AdeG pair (lane 5). Under these conditions, 2.79% of the fragment with 2-OH-AdeG was cleaved by five units of the MutY glycosylase (Table 1). When the A8-OH-Gua mismatch-containing oligonucleotide was incubated with 0.1 units of MutY, 31% of the fragment was excised. Thus, the activity of MutY for 2-OH-AdeG was estimated to be less than 0.2% of that for A8-OH-Gua. The MutY protein recognizes A residues opposite G, C, 8-OH-Gua, and 8-hydroxyadenine5,6). Moreover, it was reported that MutY removed G opposite 8-OH-Gua22). Therefore, we investigated whether a strand with A or G opposite 2-OH-Ade was cleaved by the MutY protein. The MutY protein cleaved the strand with A opposite 8-OH-Gua very efficiently. The cleavage of the DNA fragment containing G instead of A opposite 8-OH-Gua was about 1/8 as efficient as that of A8-OH-Gua (Table 1). On the other hand, MutY cleaved the strand with A opposite 2-OH-Ade less efficiently than G8-OH-Gua, and the excision of the G-strand of the G2-OH-Ade duplex was rarely detected (Table 1). Therefore, the DNA fragments containing 2-OH-Ade appeared to be very poor substrates of the MutY protein. This is in contrast to the situation of the mammalian homologue, MYH23). We added an unlabeled 2-OH-AdeG-containing fragment (10-fold excess) to a MutY reac 5 U 0.1 U *A GOH T A G C T G GOH *A *G *A *G ND ND tion mixture containing the labeled AG fragment. We observed that inhibition due to an excess amount of the 2-OH-AdeG fragment was similar to that by the AT fragment (data not shown). Thus, the affinity of the MutY protein for the 2-OH-AdeG fragment was weak. Next, oligodeoxyribonucleotides containing base pairs involving 2-OH-Ade were incubated with MutM. As controls, oligonucleotides containing 8-OH-GuaC and AT pairs were also treated. The cleavage of DNA fragments with 2-OH-Ade by MutM was not detected, irrespective of the base in the complementary strand, although MutM cleaved the oligonucleotide with 8-OHGua efficiently (data not shown). Thus, the MutM protein is not a 2-OH-Ade glycosylase. It was shown that 8-OH-Gua adopts the syn conformation to form the A (anti)8-OH-Gua (syn) base pair in duplex DNA24,25). On the other hand, the formation of the A (anti)G (anti) and AH+ (anti)G (syn) pairs (AH+ represents a protonated A base) was reported26). The MutY protein may re (...truncated)