Escherichia coli nucleoside diphosphate kinase does not act as a uracil-processing DNA repair nuclease
Escherichia coli nucleoside diphosphate kinase does not act as a uracil-processing DNA repair nuclease
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DOI：
10.1073/pnas.0401031101
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发表时间：
2004-04-27
影响因子：
11.1
通讯作者：
Mosbaugh, DW
中科院分区：
文献类型：
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作者：
Bennett, SE;Chen, CY;Mosbaugh, DW
大肠杆菌核苷二磷酸激酶(NDK)催化从同源二磷酸前体合成依赖于ATP的核糖核苷三磷酸。最近，NCLK多肽被报道为一种多功能碱基切除修复核酸酶，通过依次作为尿嘧啶-DNA糖基酶抑制蛋白(UGI)敏感的尿嘧啶-DNA糖基酶、无嘌呤/脱嘧啶裂解酶和T-磷酸二酯酶来处理DNA中的尿嘧啶残基[Postel，E.H.&Abramczyk，B.M.(2003)Proc.娜塔莉。阿卡德科学公司。USA100,13247-13252]。在这里，我们证明了大肠杆菌NDK多肽缺乏可检测到的尿嘧啶-DNA糖基酶活性，因此不能作为尿嘧啶处理的DNA修复核酸酶。这一发现基于以下观察：(I)尿嘧啶-DNA糖基酶活性与NDK活性不一致；(Ii)从E.coliung(-)细胞中纯化的NDK没有检测到尿嘧啶-DNA糖基酶活性；(Iii)Nclk未能与紧密结合E.coliuracil-DNA糖基酶(Ung)的UGi-Sepharose亲和柱结合。综上所述，这些观察表明，E.ColiNDK多肽不具有固有的尿嘧啶-DNA糖基化酶活性。
Escherichia coli nucleoside diphosphate kinase (Ndk) catalyzes ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the cognate diphosphate precursor. Recently, the Nclk polypeptide was reported to be a multifunctional base excision repair nuclease that processed uracil residues in DNA by acting sequentially as a uracil-DNA glycosylase inhibitor protein (Ugi)-sensitive uracil-DNA glycosylase, an apurinic/apyrimidinic-lyase, and a T-phosphodiesterase [Postel, E. H. & Abramczyk, B. M. (2003) Proc. Natl. Acad Sci. USA 100, 13247-13252]. Here we demonstrate that the E. coli Ndk polypeptide lacked detectable uracil-DNA glycosylase activity and, hence, was incapable of acting as a uracil-processing DNA repair nuclease. This finding was based on the following observations: (i) uracil-DNA glycosylase activity did not copurify with Ndk activity; (ii) Ndk purified from E. coli ung(-) cells showed no detectable uracil-DNA glycosylase activity; and (iii) Nclk failed to bind to a Ugi-Sepharose affinity column that tightly bound E. coli uracil-DNA glycosylase (Ung). Collectively, these observations demonstrate that the E. coli Ndk polypeptide does not possess inherent uracil-DNA glycosylase activity.