Yanofsky S D, Love R, McClarin J A, Rosenberg J M, Boyer H W, Greene P J
Department of Biochemistry and Biophysics, University of California at San Francisco 94143.
Proteins. 1987;2(4):273-82. doi: 10.1002/prot.340020403.
EcoRI endonuclease mutants were isolated in a methylase-deficient background following in vitro hydroxylamine mutagenesis of plasmid pKG2 (Kuhn et al.: Gene 44:253-263, 1986). Mutants which survived high-level endonuclease expression (IPTG induction) were termed null mutants. Sixty-two of 121 null mutants tested by Western blot contained normal levels of endonuclease cross-reacting protein. The complete endonuclease gene was sequenced for 27 null mutants. This group was found to consist of 20 single base-change missense mutations, 6 double mutations, and 1 triple mutation. Ten of the 20 single mutations were clustered between residues 139 and 144. When examined with respect to the structure of the EcoRI-DNA complex (McClarin et al.: Science 234:1526-1541, 1986), these alterations were found to fall predominantly into two classes: substitutions at the protein-DNA interface or substitutions at the protein-protein (dimer) interface. Protein from several of the mutants was purified and sized by using HPLC. Wild-type EcoRI endonuclease and protein from three of the DNA interface mutations (Ala139----Thr, Gly140----Ser, Arg203----Gln) appeared to be dimeric, while protein from subunit interface mutations (Glu144----Lys, Glu152----Lys, Gly210----Arg) migrated as monomers.
在对质粒pKG2进行体外羟胺诱变后，在甲基化酶缺陷的背景下分离出了EcoRI核酸内切酶突变体（库恩等人：《基因》44:253 - 263，1986年）。在高水平核酸内切酶表达（IPTG诱导）下存活的突变体被称为无效突变体。通过蛋白质印迹法检测的121个无效突变体中，有62个含有正常水平的核酸内切酶交叉反应蛋白。对27个无效突变体的完整核酸内切酶基因进行了测序。发现该组由20个单碱基变化错义突变、6个双突变和1个三突变组成。20个单突变中有10个集中在139至144位氨基酸残基之间。当根据EcoRI - DNA复合物的结构进行检查时（麦克拉林等人：《科学》234:1526 - 1541，1986年），发现这些改变主要分为两类：蛋白质 - DNA界面的取代或蛋白质 - 蛋白质（二聚体）界面的取代。通过高效液相色谱法对几种突变体的蛋白质进行了纯化和大小测定。野生型EcoRI核酸内切酶以及来自三个DNA界面突变（丙氨酸139→苏氨酸、甘氨酸140→丝氨酸、精氨酸203→谷氨酰胺）的蛋白质似乎是二聚体，而来自亚基界面突变（谷氨酸144→赖氨酸、谷氨酸152→赖氨酸、甘氨酸210→精氨酸）的蛋白质以单体形式迁移。
