A novel monofunctional DNA glycosylase activity against thymine glycol in mouse cell nuclei
A novel monofunctional DNA glycosylase activity against thymine glycol in mouse cell nuclei
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DOI：
10.1269/jrr.07100
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发表时间：
2008-05-01
影响因子：
2
通讯作者：
Kubo, Kihei
中科院分区：
文献类型：
--
作者：
Yamamoto, Ryohei;Akiyama, Miyuki;Kubo, Kihei
活性氧不断氧化 DNA 碱基并威胁遗传完整性。胸腺嘧啶乙二醇（TG）是代表性的氧化产物之一，主要通过碱基切除修复（BER）进行修复。在大肠杆菌中，已知核酸内切酶 III (Nth) 和核酸内切酶 VIII (Nei) 能够主动从 DNA 中去除 TG，并且同源物在各种生物体中都得到很好的保守。这些是双功能糖基化酶，也与脱嘌呤/脱嘧啶 (AP) 裂解酶活性相关。在本研究中，单功能 TG-DNA 糖基化酶活性被证明是某些小鼠组织中存在的主要核活性之一。通过结合高渗萃取和柱色谱，我们成功地将新活性与大多数双功能活性分离。由于据报道mNTH1可能不是主要的核活性，因此单功能糖基化酶活性与mNElL1一起可能是小鼠细胞核中主要的TG-DNA糖基化酶。发现单功能活性的最佳反应条件为pH 7-8和100-150 mM KCl，并且该活性对20 mM EDTA具有抵抗力。在脾脏和胃中检测到高单功能活性，而肝脏中的水平显着较低，这表明单功能活性的贡献在器官之间存在差异。
Reactive oxygen species continuously oxidize DNA bases and threaten the genetic integrity. Thymine glycol (TG), one of the representative oxidized products, is repaired mainly by base excision repair (BER). In Escherichia coli, endonuclease III (Nth) and endonuclease VIII (Nei) are known to actively remove TG from DNA, and the homologs are well conserved in various organisms. These are bifunctional glycosylases, also associated with apurinic/apyrimidinic (AP) lyase activity. In the present study, a monofunctional TG-DNA glycosylase activity is shown to be one of the predominant nuclear activities present in some mouse tissues. By combining hypertonic extraction and column chromatography, we successfully separated the novel activity from majority of the bifunctional activities. Since it has been reported that mNTH1 may not be a dominant nuclear activity, the monofunctional glycosylase activity, together with mNElL1, may be the major TG-DNA glycosylases in the mouse nucleus. The optimal reaction conditions for the monofunctional activity were found to be pH 7-8 and 100-150 mM KCl, and the activity was resistant to 20 mM EDTA. High monofunctional activity was detected in the spleen and stomach, while the level was significantly lower in the liver, suggesting that the contribution of the monofunctional activity is variable among organs.