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
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
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DOI：
10.1093/nar/28.6.1355
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发表时间：
2000-03-15
影响因子：
14.9
通讯作者：
Nakabeppu, Y
中科院分区：
文献类型：
--
作者：
Ohtsubo, T;Nishioka, K;Nakabeppu, Y
在Jurkat细胞的核提取物中检测到在双链寡核苷酸中的2-羟基腺嘌呤（2-OH-A）处引入碱不稳定位点的酶活性。该活性与针对腺嘌呤与鸟嘌呤和8-氧代-7，8-二氢鸟嘌呤（8-oxoG）配对的活性共洗脱，作为对应于凝胶过滤色谱上55 kDa分子量的单峰。然后进行进一步的共纯化，Western印迹显示这些活性也与52 kDa多肽共纯化，所述多肽与针对人MYH的抗体（抗hMYH）反应。重组hMYH具有与部分纯化的酶基本相似的活性，因此，hMYH可能同时具有腺嘌呤和2-OH-A DNA糖基化酶活性。在Jurkat细胞的核提取物中，检测到52 kDa多肽和少量53 kDa多肽，而在线粒体提取物中，使用抗hMYH检测到57 kDa多肽。通过扩增hMYH cDNA的5 '端区域，鉴定了10种形式的hMYH转录物，并将其分为三种类型，每种类型都具有独特的5'端序列。这些hMYH转录物可能编码多种真实的hMYH多肽，包括在Jurkat细胞中检测到的52、53和57 kDa多肽。
An enzyme activity introducing an alkali-labile site at 2-hydroxyadenine (2-OH-A) in double-stranded oligo-nucleotides 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.