Ghosh Kaushik, Lau Chi Kong, Guo Feng, Segall Anca M, Van Duyne Gregory D
Department of Biochemistry & Biophysics and Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 2005 Mar 4;280(9):8290-9. doi: 10.1074/jbc.M411668200. Epub 2004 Dec 8.
Cre recombinase is a prototypical member of the tyrosine recombinase family of site-specific recombinases. Members of this family of enzymes catalyze recombination between specific DNA sequences by cleaving and exchanging one pair of strands between the two substrate sites to form a 4-way Holliday junction (HJ) intermediate and then resolve the HJ intermediate to recombinant products by a second round of strand exchanges. Recently, hexapeptide inhibitors have been described that are capable of blocking the second strand exchange step in the tyrosine recombinase recombination pathway, leading to an accumulation of the HJ intermediate. These peptides are active in the lambda-integrase, Cre recombinase, and Flp recombinase systems and are potentially important tools for both in vitro mechanistic studies and as in vivo probes of cellular function. Here we present biochemical and crystallographic data that support a model where the peptide inhibitor binds in the center of the recombinase-bound DNA junction and interacts with solvent-exposed bases near the junction branch point. Peptide binding induces large conformational changes in the DNA strands of the HJ intermediate, which affect the active site geometries in the recombinase subunits.
Cre重组酶是位点特异性重组酶的酪氨酸重组酶家族的典型成员。该酶家族的成员通过在两个底物位点之间切割并交换一对链来催化特定DNA序列之间的重组，形成一个4向霍利迪连接体（HJ）中间体，然后通过第二轮链交换将HJ中间体解析为重组产物。最近，已经描述了能够阻断酪氨酸重组酶重组途径中第二条链交换步骤的六肽抑制剂，导致HJ中间体的积累。这些肽在λ整合酶、Cre重组酶和Flp重组酶系统中具有活性，并且对于体外机制研究和作为细胞功能的体内探针都是潜在的重要工具。在这里，我们展示了生化和晶体学数据，这些数据支持了一个模型，即肽抑制剂结合在重组酶结合的DNA连接体的中心，并与连接体分支点附近暴露于溶剂的碱基相互作用。肽结合诱导HJ中间体的DNA链发生大的构象变化，这影响了重组酶亚基中的活性位点几何形状。
