Allawi Hatim T, Kaiser Michael W, Onufriev Alexey V, Ma Wu-Po, Brogaard Andrew E, Case David A, Neri Bruce P, Lyamichev Victor I
Third Wave Technologies, Inc., 502 S Rosa Road, Madison, WI 53719, USA.
J Mol Biol. 2003 May 2;328(3):537-54. doi: 10.1016/s0022-2836(03)00351-6.
Structure-specific 5' nucleases play an important role in DNA replication and repair uniquely recognizing an overlap flap DNA substrate and processing it into a DNA nick. However, in the absence of a high-resolution structure of the enzyme/DNA complex, the mechanism underlying this recognition and substrate specificity, which is key to the enzyme's function, remains unclear. Here, we propose a three-dimensional model of the structure-specific 5' flap endonuclease from Pyrococcus furiosus in its complex with DNA. The model is based on the known X-ray structure of the enzyme and a variety of biochemical and molecular dynamics (MD) data utilized in the form of distance restraints between the enzyme and the DNA. Contacts between the 5' flap endonuclease and the sugar-phosphate backbone of the overlap flap substrate were identified using enzyme activity assays on substrates with methylphosphonate or 2'-O-methyl substitutions. The enzyme footprint extends two to four base-pairs upstream and eight to nine base-pairs downstream of the cleavage site, thus covering 10-13 base-pairs of duplex DNA. The footprint data are consistent with a model in which the substrate is bound in the DNA-binding groove such that the downstream duplex interacts with the helix-hairpin-helix motif of the enzyme. MD simulations to identify the substrate orientation in this model are consistent with the results of the enzyme activity assays on the methylphosphonate and 2'-O-methyl-modified substrates. To further refine the model, 5' flap endonuclease variants with alanine point substitutions at amino acid residues expected to contact phosphates in the substrate and one deletion mutant were tested in enzyme activity assays on the methylphosphonate-modified substrates. Changes in the enzyme footprint observed for two point mutants, R64A and R94A, and for the deletion mutant in the enzyme's beta(A)/beta(B) region, were interpreted as being the result of specific interactions in the enzyme/DNA complex and were used as distance restraints in MD simulations. The final structure suggests that the substrate's 5' flap interacts with the enzyme's helical arch and that the helix-hairpin-helix motif interacts with the template strand in the downstream duplex eight base-pairs from the cleavage site. This model suggests specific interactions between the 3' end of the upstream oligonucleotide and the enzyme. The proposed structure presents the first detailed description of substrate recognition by structure-specific 5' nucleases.
结构特异性5'核酸酶在DNA复制和修复过程中发挥着重要作用，它能独特地识别重叠瓣状DNA底物，并将其加工成DNA切口。然而，由于缺乏酶/DNA复合物的高分辨率结构，这种识别和底物特异性的机制（这是酶功能的关键）仍不清楚。在此，我们提出了嗜热栖热菌结构特异性5'瓣状内切核酸酶与DNA复合物的三维模型。该模型基于该酶已知的X射线结构以及以酶与DNA之间的距离约束形式使用的各种生化和分子动力学（MD）数据。通过对含有甲基膦酸酯或2'-O-甲基取代的底物进行酶活性测定，确定了5'瓣状内切核酸酶与重叠瓣状底物的糖磷酸骨架之间的接触。酶足迹在切割位点上游延伸两到四个碱基对，在下游延伸八到九个碱基对，从而覆盖10 - 13个碱基对的双链DNA。足迹数据与一个模型一致，在该模型中底物结合在DNA结合凹槽中，使得下游双链与酶的螺旋-发夹-螺旋基序相互作用。在该模型中识别底物取向的MD模拟与对甲基膦酸酯和2'-O-甲基修饰底物的酶活性测定结果一致。为了进一步完善模型，在对甲基膦酸酯修饰底物的酶活性测定中测试了在预期与底物中的磷酸基团接触的氨基酸残基处具有丙氨酸点突变的5'瓣状内切核酸酶变体和一个缺失突变体。观察到的两个点突变体R64A和R94A以及酶的β(A)/β(B)区域中的缺失突变体在酶足迹上的变化被解释为酶/DNA复合物中特定相互作用的结果，并在MD模拟中用作距离约束。最终结构表明底物的5'瓣与酶的螺旋拱相互作用，并且螺旋-发夹-螺旋基序与切割位点下游八个碱基对处的下游双链中的模板链相互作用。该模型表明上游寡核苷酸的3'端与酶之间存在特定相互作用。所提出的结构首次详细描述了结构特异性5'核酸酶对底物的识别。
