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.
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.
Ohtsubo
T
T
Department of Biochemistry, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
Nishioka
K
K
Imaiso
Y
Y
Iwai
S
S
Shimokawa
H
H
Oda
H
H
Fujiwara
T
T
Nakabeppu
Y
Y
D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002457:Cell Extracts; D002467:Cell Nucleus / Q000201:enzymology; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D045647:DNA Glycosylases; D004260:DNA Repair / Q000235:genetics*; D020869:Gene Expression Profiling; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019169:Jurkat Cells; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D002457:Cell Extracts; D007527:Isoenzymes; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; C008183:isoguanine; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D000225:Adenine