Pang Zhenhua, Chen Ray, Manna Dipankar, Higgins N Patrick
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 35294, USA.
J Bacteriol. 2005 Nov;187(22):7773-83. doi: 10.1128/JB.187.22.7773-7783.2005.
When a mutation in an essential gene shows a temperature-sensitive phenotype, one usually assumes that the protein is inactive at nonpermissive temperature. DNA gyrase is an essential bacterial enzyme composed of two subunits, GyrA and GyrB. The gyrB652 mutation results from a single base change that substitutes a serine residue for arginine 436 (R436-S) in the GyrB protein. At 42 degrees C, strains with the gyrB652 allele stop DNA replication, and at 37 degrees C, such strains grow but have RecA-dependent SOS induction and show constitutive RecBCD-dependent DNA degradation. Surprisingly, the GyrB652 protein is not inactive at 42 degrees C in vivo or in vitro and it doesn't directly produce breaks in chromosomal DNA. Rather, this mutant has a low k(cat) compared to wild-type GyrB subunit. With more than twice the normal mean number of supercoil domains, this gyrase hypomorph is prone to fork collapse and topological chaos near the terminus of DNA replication.
当一个必需基因中的突变表现出温度敏感型表型时，人们通常认为该蛋白质在非允许温度下是无活性的。DNA促旋酶是一种由GyrA和GyrB两个亚基组成的必需细菌酶。gyrB652突变源于单个碱基变化，该变化使GyrB蛋白中的精氨酸436（R436）被丝氨酸残基取代（R436-S）。在42℃时，带有gyrB652等位基因的菌株停止DNA复制，而在37℃时，此类菌株能够生长，但具有RecA依赖的SOS诱导，并表现出组成型RecBCD依赖的DNA降解。令人惊讶的是，GyrB652蛋白在体内或体外42℃时并非无活性，并且它不会直接导致染色体DNA断裂。相反，与野生型GyrB亚基相比，这种突变体的催化常数（k(cat)）较低。由于超螺旋结构域的平均数量比正常情况多两倍以上，这种促旋酶功能减弱型突变体在DNA复制终点附近容易发生复制叉坍塌和拓扑混乱。
