Schmiedel Ramona, Kuettner E Bartholomeus, Keim Antje, Sträter Norbert, Greiner-Stöffele Thomas
Institute of Biochemistry, Faculty of Biology, Pharmacy and Psychology, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
DNA Repair (Amst). 2009 Feb 1;8(2):219-31. doi: 10.1016/j.dnarep.2008.10.009. Epub 2008 Dec 5.
The major AP endonuclease in Escherichia coli Exonuclease III (ExoIII) is frequently used in gene technology due to its strong exonucleolytic activity. A thermostabilized variant of ExoIII or a homologous enzyme from thermophilic organisms could be most useful for further applications. For this purpose we characterized a nuclease from the hyperthermophilic archaeon Archaeoglobus fulgidus (Af_Exo), which shares 33% overall sequence identity and 55% similarity to ExoIII. The gene coding for this thermostable enzyme was cloned and expressed in E. coli. The purified protein shows a strong Mg(2+)-dependent nicking activity at AP-sites, nicking of undamaged double-stranded (ds) DNA and a weak exonucleolytic activity. A V217G variant of the enzyme was crystallized with decamer ds-DNA molecule, and the three-dimensional structure was determined to 1.7A resolution. Besides our goal to find or produce a thermostable exonuclease, the structural and catalytic data of Af_Exo and a series of mutant proteins, based on the crystal structure, provide new insight into the mechanism of abasic site recognition and repair. Each of the hydrophobic residues Phe 200, Trp 215 and Val 217, forming a binding pocket for the abasic deoxyribose in Af_Exo, were mutated to glycine or serine. By expanding the size of the binding pocket the unspecific endonucleolytic activity is increased. Thus, size and flexibility of the mostly hydrophobic binding pocket have a significant influence on AP-site specificity. We suggest that its tight fitting to the flipped-out deoxyribose allows for a preferred competent binding of abasic sites. In a larger or more flexible pocket however, intact nucleotides more easily bind in a catalytically competent conformation, resulting in loss of specificity. Moreover, with mutations of Phe 200 and Trp 215 we induced a strong exonucleolytic activity on undamaged DNA.
大肠杆菌中的主要 AP 核酸内切酶核酸外切酶 III（ExoIII）因其强大的核酸外切酶活性而在基因技术中经常被使用。ExoIII 的热稳定变体或来自嗜热生物的同源酶对于进一步的应用可能最为有用。为此，我们对来自超嗜热古菌嗜热栖热菌（Af_Exo）的一种核酸酶进行了表征，它与 ExoIII 的总体序列同一性为 33%，相似性为 55%。编码这种热稳定酶的基因被克隆并在大肠杆菌中表达。纯化后的蛋白质在 AP 位点表现出强烈的 Mg(2+) 依赖性切口活性、对未受损双链（ds）DNA 的切口活性以及较弱的核酸外切酶活性。该酶的 V217G 变体与十聚体 ds - DNA 分子结晶，并确定了分辨率为 1.7Å 的三维结构。除了我们寻找或生产热稳定核酸外切酶的目标外，基于晶体结构的 Af_Exo 和一系列突变蛋白的结构及催化数据，为无碱基位点识别和修复机制提供了新的见解。在 Af_Exo 中形成无碱基脱氧核糖结合口袋的每个疏水残基苯丙氨酸 200、色氨酸 215 和缬氨酸 217 都被突变为甘氨酸或丝氨酸。通过扩大结合口袋的大小，非特异性内切核酸酶活性增加。因此，主要为疏水的结合口袋的大小和灵活性对 AP 位点特异性有显著影响。我们认为它与翻转出的脱氧核糖紧密契合，使得无碱基位点能够优先进行有效结合。然而，在更大或更灵活的口袋中，完整的核苷酸更容易以催化活性构象结合，导致特异性丧失。此外通过苯丙氨酸 200 和色氨酸 215 的突变，我们在未受损 DNA 上诱导出了强烈的核酸外切酶活性。
