Structural analysis of endonuclease-DNA interaction and alteration of substrate specificity

核酸内切酶-DNA 相互作用的结构分析和底物特异性的改变

• 批准号: 16380061
• 负责人: KITA Keiko
• 金额: $ 9.79万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16380061/
• 关键词: Restriction endonuclease; Protein engineering; Alteration of substrate specificity; DNA-binding protein; Crystal structure analysis; 制限修飾系; エンドヌクレアーゼ; 結晶化; 部位特異的変異; 金属イオン; 界面活性剤; 転写制御因子; DNA認識機構; 構造生物学

1.EcoO109I isolated from E. coli H709c recognizes the degenerate DNA sequence PuGGNCCPy. In the EcoO109I-DNA co-crystal structure, the K173 and the K74 residues locate in the vicinity of the degenerate base G in the recognition sequence and G outside of the recognition sequence, respectively. To investigate the substrate specificity conferred by these residues and to alter EcoO109I specificity, variants K173R and K74R were purified and their cleavage activity determined. K173R showed less than 0.1% of the wild-type activity, in contrast, K74R showed almost equal activity. In the presence of 2.5 mM Mg^<2+>, 50% glycerol or 25% DMSO, wild-type EcoO109I showed relaxed substrate specificity or so-called star activity, however, K74R variant cleaved star sites poorly. It was clarified that the recognition specificity of K74R variant was more accurate than that of the wild-type enzyme. In order to understand the selectivity of sequence recognition of EcoO109I, K74R variant-DNA co-crystals were obtained and will be submitted to X-ray diffraction analysis.2.EcoT38I isolated from E. coli TH38 recognizes the degenerate DNA sequence GPuGCPyC and cleaves the DNA in the presence of Mg^<2+>, Mn^<2+> and Co^<2+>, but not in the presence of Ca^<2+>. However, Ca^<2+> showed the effect which promoted the cleavage activity by Mn^<2+>, while the cleavage activity by Mg^<2+> was suppressed. This suggests the two metal reaction mechanism. EcoT38I also showed relaxed substrate specificity or so-called star activity in the presence of low concentration of Mg^<2+> or Mn^<2+>. Ca^<2+> suppressed star activities induced by Mg^<2+> or Mn^<2+>. In order to understand the mechanism of recognition and cleavage of DNA, crystals of EcoT38I and its complex with DNA were obtained and were submitted to X-ray diffraction analysis. Diffraction data were collected to 2.1 Å and 3.3Å resolution, respectively.

1.从大肠杆菌H709c分离到的EcoO109I识别简并的DNA序列PuGGNCCPy。在EcoO109I-DNA共晶结构中，K173和K74残基分别位于识别序列中的简并碱基G附近和识别序列外的G附近。为了研究这些残基赋予的底物专一性和改变EcoO109I的专一性，我们纯化了突变体K173R和K74R，并测定了它们的切割活性。K173R的活性低于野生型的0.1%，而K74R的活性几乎与野生型相当。在2.5 mM镁离子、50%甘油或25%DMSO存在下，野生型EcoO109I表现出松弛的底物专一性或所谓的STAR活性，而K74R变异体切割STAR活性较差。结果表明，K74R变异体比野生型酶具有更高的识别特异性。为了了解EcoO109I的序列识别选择性，我们获得了K74R变异DNA共晶体，并将其提交给X射线衍射分析。2.从大肠杆菌TH38中分离的EcoT38I识别简并的DNA序列GPuGCPyC，并在Mg^&lt；2+&gt；，Mn^&lt；2+&gt；和Co^&lt；2+&gt；存在下切割DNA，而在Ca^&lt；2+&gt；存在下不切割DNA。而Ca~(2+)对Mn~(2+)的切割活性有促进作用，而对Mg~(2+)的切割活性有抑制作用。这表明这是两种金属反应的机理。EcoT38I在低浓度的镁离子或锰离子存在下也表现出松弛的底物专一性或所谓的STAR活性。Ca^&lt；2+&gt；抑制了Mg^&lt；2+&gt；或Mn^&lt；2+&gt；诱导的星体活动。为了了解EcoT38I与DNA的识别和切割机理，我们获得了EcoT38I及其与DNA的络合物的晶体，并对其进行了X射线衍射分析。分别采集了2.1Å和3.3Å分辨率的衍射数据。