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. 2000 Mar 15;28(6):1355-64.
doi: 10.1093/nar/28.6.1355.

Identification of human MutY homolog (hMYH) as a repair enzyme for 2-hydroxyadenine in DNA and detection of multiple forms of hMYH located in nuclei and mitochondria

T Ohtsubo  1 , K Nishioka, Y Imaiso, S Iwai, H Shimokawa, H Oda, T Fujiwara, Y Nakabeppu
Affiliations

Identification of human MutY homolog (hMYH) as a repair enzyme for 2-hydroxyadenine in DNA and detection of multiple forms of hMYH located in nuclei and mitochondria

T Ohtsubo et al. Nucleic Acids Res. .

Erratum in

Abstract

An enzyme activity introducing an alkali-labile site at 2-hydroxyadenine (2-OH-A) in double-stranded oligonucleotides was detected in nuclear extracts of Jurkat cells. This activity co-eluted with activities toward adenine paired with guanine and 8-oxo-7,8-dihydroguanine (8-oxoG) as a single peak corresponding to a 55 kDa molecular mass on gel filtration chromatography. Further co-purification was then done. Western blotting revealed that these activities also co-purified with a 52 kDa polypeptide which reacted with antibodies against human MYH (anti-hMYH). Recombinant hMYH has essentially similar activities to the partially purified enzyme. Thus, hMYH is likely to possess both adenine and 2-OH-A DNA glycosylase activities. In nuclear extracts from Jurkat cells, a 52 kDa polypeptide was detected with a small amount of 53 kDa polypeptide, while in mitochondrial extracts a 57 kDa polypeptide was detected using anti-hMYH. With amplification of the 5'-regions of the hMYH cDNA, 10 forms of hMYH transcripts were identified and subgrouped into three types, each with a unique 5' sequence. These hMYH transcripts are likely to encode multiple authentic hMYH polypeptides including the 52, 53 and 57 kDa polypeptides detected in Jurkat cells.

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Figures

Figure 1
Figure 1
Co-purification of repair activities for 2-OH-A and adenine paired with guanine or 8-oxoG. Nuclear extracts prepared from Jurkat cells were applied to successive chromatographies, as summarized in Table 1. Each fraction from gel filtration chromatography was incubated with various double-stranded oligonucleotides containing the abnormal base pair shown in the figure and nicking activities were determined as described in Materials and Methods. * indicates a strand labeled at its 5′-end with FAM. AO, 2-OH-A; GO, 8-oxoG.
Figure 2
Figure 2
Authentic hMYH possesses repair activity for 2-OH-A and adenine paired with guanine or 8-oxoG. The RESOURCE S fraction was subjected to nicking assay for various double-stranded oligonucleotides (A). The active fractions were subjected to SDS–PAGE and then the gel was stained with silver (B) or was further analyzed by western blotting, using anti-hMYH which had been pre-adsorbed with TrpE–Sepharose (C) or TrpE-hMYH–Sepharose (D).
Figure 3
Figure 3
Characterization of the enzymatic properties of partially purified hMYH. (A) Substrate specificity. A partially purified fraction of hMYH (the RESOURCE S fraction) was incubated with various double-stranded oligonucleotides. Reaction products were fractionated on a 6% LongRanger gel containing 7 M urea, after mild alikaline treatment. Relative amounts of cleaved product of each substrate to that of A:G substrate are shown. 19-P, FAM-labeled control oligonucleotide corresponding to the cleaved product at the 5′-side of adenine or 2-OH-A, with 3′-phosphate; 20-OH, FAM-labeled control oligonucleotide corresponding to the cleaved product at the 3′-side of adenine or 2-OH-A, without 3′-phosphate. (B) NaOH-dependent nicking of oligonucleotide containing adenine paired with 8-oxoG by partially purified hMYH fraction. Various amounts of the enzyme fraction were incubated with double-stranded oligonucleotides containing adenine paired with 8-oxoG and the mixture was fractionated with or without NaOH treatment. (C) Fluorescence intensities of bands corresponding to the cleavage products produced by hMYH were obtained from data in (B) and plotted. (D) AP lyase activity in partially purified hMYH fraction. Double-stranded oligonucleotides containing uracil paired with guanine were first treated with uracil DNA glycosylase, then incubated with or without the enzyme fraction. The products were fractionated with or without NaOH treatment.
Figure 4
Figure 4
Recombinant hMYH possesses DNA repair activity for 2-OH-A and adenine paired with guanine or 8-oxoG. (A) Western blotting. Extracts (5 µl) prepared from E.coli cells carrying plasmid pXC35:14-3-3 (lane C) or pXC35:hMYHα3-2 (lane Y) were subjected to western blot analysis, using anti-hMYH. (B) DNA repair activities of recombinant hMYH. Various double-stranded oligonucleotides (150 fmol/15 µl reaction) were incubated with extracts (5 µl) prepared from E.coli cells carrying plasmid pXC35:14-3-3 (lane C) or pXC35:hMYHα3-2 (lane Y) for 30 min at 37°C, then 3 µl of 1 N NaOH was added, followed by heating at 95°C for 10 min. Then the mixture was fractionated on an 8% LongRanger gel containing 7 M urea. Relative amounts of cleaved products of each substrate produced by hMYH to that of A:GO substrate are shown.
Figure 5
Figure 5
5. Immunological detection of authentic hMYH in human cells. (A) Western blotting. In vitro translation products of the pT7Blue vector (V), pT7Blue:hMYHα3 (α3) and pT7Blue:hMYHα3-2 (α3-2), and isolated nuclei (N) and mitochondria (Mt) (equivalent to 50 µg of protein) from Jurkat cells and partially purified hMYH in the RESOURCE S fraction (RS) were subjected to western blot analysis, using anti-hMYH. (B) Submitochondrial localization of hMYH, determined by electron microscopic immunocytochemistry. After mitochondria had been isolated from Jurkat cells, thin sections (~0.1 µm) were prepared for electron microscopic immunocytochemistry with anti-hMYH preadsorbed with TrpE–Sepharose or with TrpE-hMYH–Sepharose in combination with protein A–gold. Bars indicate 0.2 µm.
Figure 6
Figure 6
6. Multiple forms of hMYH transcripts and predicted polypeptides. (A) Sequences of 5′-regions (exons 1–3) of 10 different forms of hMYH cDNA. Sequences corresponding to the reported exons 2 and 3 are shown in box (18). Two putative initiation codons, ATG in exons 1 and 2, are shown in italic. (B) Predicted polypeptides encoded by the various hMYH transcripts. * indicates a methionine residue. The gray box indicates the mitochondrial _targeting signal (19,56) and the hatched box indicates the 11 amino acid insertion.
Figure 7
Figure 7
Expression of hMYH mRNAs in various human tissues and cell lines. (A) Northern blot analysis. Total RNAs (16 µg each) extracted from various human tissues and Jurkat and HeLa S3 cells were electrophoresed, transferred to nitrocellulose membrane and probed with 32P-labeled hMYH cDNA. (B) RT–PCR analysis. cDNAs were synthesized from total RNA prepared from HeLa S3 cells, fetal brain, adult brain and kidney, using oligo(dT)18 primer. The cDNAs were amplified using a common 3′ primer (MYH3′-4) and a 5′ primer specific for each type of hMYH mRNA (types 5′α, 5′β and 5′γ). PCR products were analyzed by 1.5% agarose gel electrophoresis. The presence of primers in the reaction mixture is shown by +.
Figure 8
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) and those for hMTH1 and MutT are based on the data of Fujii et al. (45). Residues conserved among more than three proteins are shown in a yellow box and residues conserved only between MutT and hMTH1 or hMYH and MutY are shown in a blue or green box, respectively. Residues conserved only between hMTH1 and hMYH, which may be involved in 2-OH-A recognition, are shown in a red box.
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