Characterization of an endonuclease IV 3′-5′ exonuclease activity
Characterization of an endonuclease IV 3′-5′ exonuclease activity
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DOI：
10.1074/jbc.m210750200
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发表时间：
2003-01-31
影响因子：
4.8
通讯作者：
McCarthy, TV
中科院分区：
文献类型：
--
作者：
Kerins, SM;Collins, R;McCarthy, TV
先前对大肠杆菌核酸内切酶 IV 的表征表明，该酶在无嘌呤/无嘧啶位点特异性切割 DNA 主链，并去除 3' DNA 封闭基团。相比之下，与主要的无嘌呤/脱嘧啶核酸内切酶核酸外切酶 III 不同，核酸外切酶 IV 的相关核酸外切酶活性可以忽略不计。在这里，我们报告核酸内切酶 IV 确实具有内在的 3'-5' 核酸外切酶活性。在大肠杆菌和远缘嗜热菌海栖热袍菌的核酸内切酶 IV 蛋白的纯化制剂中检测到了该活性；它在不同的色谱条件下与两种酶共洗脱。在大肠杆菌过表达系统中诱导任一核酸内切酶IV都会导致核酸外切酶活性的诱导，并且大肠杆菌外切核酸酶活性与核酸内切酶IV A-P核酸内切酶活性具有相似的热稳定性。核酸外切酶活性的表征表明，其在底物上的进展对离子强度、金属离子、EDTA 和还原条件敏感。具有 3' 凹进末端的底物是 3'-5' 核酸外切酶活性的优选底物。核酸内切酶IV的相对无嘌呤/脱嘧啶核酸内切酶和核酸外切酶活性的比较表明，相对核酸外切酶活性高并且可能在体内显着。
Previous characterization of Escherichia coli endonuclease IV has shown that the enzyme specifically cleaves the DNA backbone at apurinic/apyrimidinic sites and removes 3' DNA blocking groups. By contrast, and unlike the major apurinic/apyrimidinic endonuclease exonuclease III, negligible exonuclease activity has been associated with endonuclease IV. Here we report that endonuclease IV does possess an intrinsic 3'-5' exonuclease activity. The activity was detected in purified preparations of the endonuclease IV protein from E. coli and from the distantly related thermophile Thermotoga maritima; it co-eluted with both enzymes under different chromatographic conditions. Induction of either endonuclease IV in an E. coli overexpression system resulted in induction of the exonuclease activity, and the E. coli exonuclease activity had similar heat stability to the endonuclease IV A-P endonuclease activity. Characterization of the exonuclease activity showed that its progression on substrate is sensitive to ionic strength, metal ions, EDTA, and reducing conditions. Substrates with 3' recessed ends were preferred substrates for the 3'-5' exonuclease activity. Comparison of the relative apurinic/apyrimidinic endonuclease and exonuclease activity of endonuclease IV shows that the relative exonuclease activity is high and is likely to be significant in vivo.