2006 Fiscal Year Final Research Report Summary

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

Research Project

Project/Area Number	16380061
Research Category	Grant-in-Aid for Scientific Research (B)
Allocation Type	Single-year Grants
Section	一般
Research Field	Applied microbiology
Research Institution	Kyoto University
Principal Investigator	KITA Keiko Kyoto University, Dept.of Agriculture, Professor, 農学研究科, 教授 (70234226)
Co-Investigator(Kenkyū-buntansha)	SAKO Yoshihiko Kyoto University, Dept.of Agriculture, Professor, 農学研究科, 教授 (60153970) TSUGE Hideaki Tokushima Bunri University, Inst.for Health Sciences, Professor, 健康科学研究所, 教授 (40299342)
Project Period (FY)	2004 – 2006
Keywords	Restriction endonuclease / Protein engineering / Alteration of substrate specificity / DNA-binding protein / Crystal structure analysis
Research Abstract	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.