Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario N6A 5C1, Canada.
Nucleic Acids Res. 2013 May 1;41(10):5413-27. doi: 10.1093/nar/gkt186. Epub 2013 Apr 4.
The GIY-YIG nuclease domain is found within protein scaffolds that participate in diverse cellular pathways and contains a single active site that hydrolyzes DNA by a one-metal ion mechanism. GIY-YIG homing endonucleases (GIY-HEs) are two-domain proteins with N-terminal GIY-YIG nuclease domains connected to C-terminal DNA-binding and they are thought to function as monomers. Using I-BmoI as a model GIY-HE, we test mechanisms by which the single active site is used to generate a double-strand break. We show that I-BmoI is partially disordered in the absence of substrate, and that the GIY-YIG domain alone has weak affinity for DNA. Significantly, we show that I-BmoI functions as a monomer at all steps of the reaction pathway and does not transiently dimerize or use sequential transesterification reactions to cleave substrate. Our results are consistent with the I-BmoI DNA-binding domain acting as a molecular anchor to tether the GIY-YIG domain to substrate, permitting rotation of the GIY-YIG domain to sequentially nick each DNA strand. These data highlight the mechanistic differences between monomeric GIY-HEs and dimeric or tetrameric GIY-YIG restriction enzymes, and they have implications for the use of the GIY-YIG domain in genome-editing applications.
GIY-YIG 核酸内切酶结构域存在于参与多种细胞途径的蛋白质支架中，它包含一个单一的活性位点，通过单金属离子机制水解 DNA。GIY-YIG 归巢内切酶（GIY-HEs）是具有 N 端 GIY-YIG 核酸内切酶结构域和 C 端 DNA 结合结构域的二域蛋白，被认为以单体形式发挥作用。我们以 I-BmoI 作为模型 GIY-HE，测试了单个活性位点用于产生双链断裂的机制。我们表明，在没有底物的情况下，I-BmoI 部分无定形，并且 GIY-YIG 结构域本身对 DNA 的亲和力较弱。重要的是，我们表明 I-BmoI 在反应途径的所有步骤中都作为单体发挥作用，并且不会瞬时二聚化或使用连续的转酯反应来切割底物。我们的结果与 I-BmoI DNA 结合结构域作为分子锚将 GIY-YIG 结构域固定在底物上的作用一致，从而允许 GIY-YIG 结构域旋转以顺序切割每个 DNA 链。这些数据突出了单体 GIY-HEs 与二聚体或四聚体 GIY-YIG 限制酶之间的机制差异，并且对 GIY-YIG 结构域在基因组编辑应用中的使用具有影响。
