Hatfull G F, Grindley N D
Proc Natl Acad Sci U S A. 1986 Aug;83(15):5429-33. doi: 10.1073/pnas.83.15.5429.
The resolvase encoded by the transposon gamma delta mediates a site-specific recombination between the two copies of gamma delta in a cointegrate molecule to yield the final products of transposition. Several mutants of resolvase that lack recombinational activity have been isolated previously, and one of these, which has a serine-to-leucine change at position 10, we have characterized in vitro. We also have constructed a serine-to-cysteine change at position 10 by in vitro mutagenesis and have analyzed this mutant protein in vitro. We find that the cysteine-10 mutant is defective in recombinational activity but binds to the recombinational site, res, similarly to wild-type, as assayed by gel electrophoresis of the protein-DNA complexes. In contrast, the leucine-10 mutant has a specific defect in complex formation with site I, which contains the recombinational crossover point, although it can bind and provide the ancillary functions of resolvase at sites II and III. It has been shown that a phosphoserine linkage is made to the DNA during recombination; because serine-10 is absolutely conserved amongst the family of homologous site-specific recombination proteins, it is a good candidate for the active-site serine. The properties of these resolvase mutants with substitutions at position 10 are consistent with this hypothesis and suggest that serine-10 is in close contact with the DNA at site I.
转座子γδ编码的解离酶介导共整合分子中两个γδ拷贝之间的位点特异性重组，从而产生转座的最终产物。先前已分离出几种缺乏重组活性的解离酶突变体，其中一种在第10位发生了丝氨酸到亮氨酸的变化，我们已在体外对其进行了表征。我们还通过体外诱变在第10位构建了丝氨酸到半胱氨酸的变化，并在体外分析了这种突变蛋白。我们发现，通过蛋白质-DNA复合物的凝胶电泳分析，第10位半胱氨酸突变体在重组活性方面存在缺陷，但与野生型类似，能与重组位点res结合。相比之下，第10位亮氨酸突变体在与包含重组交叉点的位点I形成复合物时存在特定缺陷，尽管它能在位点II和III结合并发挥解离酶的辅助功能。研究表明，重组过程中会形成磷酸丝氨酸与DNA的连接；由于丝氨酸-10在同源位点特异性重组蛋白家族中绝对保守，它是活性位点丝氨酸的良好候选者。这些在第10位发生替代的解离酶突变体的特性与这一假设一致，并表明丝氨酸-10在位点I处与DNA紧密接触。
