Gibb Bryan, Gupta Kushol, Ghosh Kaushik, Sharp Robert, Chen James, Van Duyne Gregory D
Department of Biochemistry and Biophysics and Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
Nucleic Acids Res. 2010 Sep;38(17):5817-32. doi: 10.1093/nar/gkq384. Epub 2010 May 12.
Members of the tyrosine recombinase (YR) family of site-specific recombinases catalyze DNA rearrangements using phosphoryl transfer chemistry that is identical to that used by the type IB topoisomerases (TopIBs). To better understand the requirements for YR catalysis and the relationship between the YRs and the TopIBs, we have analyzed the in vivo and in vitro recombination activities of all substitutions of the seven active site residues in Cre recombinase. We have also determined the structure of a vanadate transition state mimic for the Cre-loxP reaction that facilitates interpretation of mutant activities and allows for a comparison with similar structures from the related topoisomerases. We find that active site residues shared by the TopIBs are most sensitive to substitution. Only two, the tyrosine nucleophile and a conserved lysine residue that activates the 5'-hydroxyl leaving group, are strictly required to achieve >5% of wild-type activity. The two conserved arginine residues each tolerate one substitution that results in modest recombination activity and the remaining three active site positions can be substituted with several alternative amino acids while retaining a significant amount of activity. The results are discussed in the context of YR and TopIB structural models and data from related YR systems.
酪氨酸重组酶（YR）家族的位点特异性重组酶成员利用与IB型拓扑异构酶（TopIBs）相同的磷酸转移化学催化DNA重排。为了更好地理解YR催化的要求以及YR与TopIBs之间的关系，我们分析了Cre重组酶中七个活性位点残基所有取代形式的体内和体外重组活性。我们还确定了Cre-loxP反应的钒酸盐过渡态模拟物的结构，这有助于解释突变体活性，并能与相关拓扑异构酶的类似结构进行比较。我们发现TopIBs共有的活性位点残基对取代最为敏感。要达到野生型活性的5%以上，严格来说仅需两个残基，即酪氨酸亲核试剂和激活5'-羟基离去基团的保守赖氨酸残基。两个保守的精氨酸残基各自能耐受一次取代，这会导致适度的重组活性，其余三个活性位点位置可用几种替代氨基酸进行取代，同时仍保留大量活性。我们将结合YR和TopIB结构模型以及来自相关YR系统的数据来讨论这些结果。
