Leschziner A E, Boocock M R, Grindley N D
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
Mol Microbiol. 1995 Mar;15(5):865-70. doi: 10.1111/j.1365-2958.1995.tb02356.x.
Site-specific recombinases of the resolvase and DNA invertase family all contain a tyrosine residue close to the N-terminus, and four residues away from a serine that has been implicated in catalysis of DNA strand breakage and reunion. To examine the role of this tyrosine in recombination, we have constructed a mutant of gamma delta resolvase in which the tyrosine (residue 6) is replaced by phenylalanine. Characterization of the Y6F mutant protein in vitro indicated that although it was highly defective in recombination, it could cleave DNA at the cross-over site, form a covalent resolvase-DNA complex and rejoin the cleaved cross-over site (usually restoring the parental site). These data rule out a direct role of the Tyr-6 hydroxyl as the nucleophile in the DNA cleavage reaction and strengthen the conclusion that this nucleophile is the nearby invariant serine residue, Ser-10. We conclude that Tyr-6 is essential for fully co-ordinated strand cleavage and exchange, but is dispensable for individual strand cleavage and religation reactions.
解离酶和DNA转化酶家族的位点特异性重组酶在靠近N端处均含有一个酪氨酸残基，且与一个丝氨酸残基相隔四个残基，该丝氨酸残基与DNA链断裂和重新连接的催化作用有关。为了研究该酪氨酸在重组中的作用，我们构建了一个γδ解离酶突变体，其中酪氨酸（第6位残基）被苯丙氨酸取代。对Y6F突变体蛋白进行的体外特性分析表明，尽管它在重组方面存在高度缺陷，但它能够在交叉位点切割DNA，形成共价解离酶-DNA复合物，并重新连接切割后的交叉位点（通常恢复亲本位点）。这些数据排除了Tyr-6羟基在DNA切割反应中作为亲核试剂的直接作用，并强化了这样的结论，即该亲核试剂是附近不变的丝氨酸残基Ser-10。我们得出结论，Tyr-6对于完全协调的链切割和交换至关重要，但对于单个链切割和重新连接反应来说并非必需。
