Chevalier B S, Monnat R J, Stoddard B L
Fred Hutchinson Cancer Research Center, University of Washington, 1100 Fairview Ave. N. A3-023, Seattle, Washington 98109, USA.
Nat Struct Biol. 2001 Apr;8(4):312-6. doi: 10.1038/86181.
Homing endonucleases, like restriction enzymes, cleave double-stranded DNA at specific target sites. The cleavage mechanism(s) utilized by LAGLIDADG endonucleases have been difficult to elucidate; their active sites are divergent, and only one low resolution cocrystal structure has been determined. Here we report two high resolution structures of the dimeric I-CreI homing endonuclease bound to DNA: a substrate complex with calcium and a product complex with magnesium. The bound metals in both complexes are verified by manganese anomalous difference maps. The active sites are positioned close together to facilitate cleavage across the DNA minor groove; each contains one metal ion bound between a conserved aspartate (Asp 20) and a single scissile phosphate. A third metal ion bridges the two active sites. This divalent cation is bound between aspartate residues from the active site of each subunit and is in simultaneous contact with the scissile phosphates of both DNA strands. A metal-bound water molecule acts as the nucleophile and is part of an extensive network of ordered water molecules that are positioned by enzyme side chains. These structures illustrate a unique variant of a two-metal endonuclease mechanism is employed by the highly divergent LAGLIDADG enzyme family.
归巢内切酶与限制性内切酶一样，能在特定的靶位点切割双链DNA。LAGLIDADG内切酶所利用的切割机制一直难以阐明；它们的活性位点各不相同，仅确定了一个低分辨率的共晶体结构。在此，我们报道了二聚体I-CreI归巢内切酶与DNA结合的两个高分辨率结构：一个与钙结合的底物复合物和一个与镁结合的产物复合物。两种复合物中结合的金属均通过锰异常差分图得到验证。活性位点靠得很近，便于在DNA小沟中进行切割；每个活性位点都含有一个结合在保守天冬氨酸（Asp 20）和单个可切割磷酸基团之间的金属离子。第三个金属离子桥接两个活性位点。这个二价阳离子结合在每个亚基活性位点的天冬氨酸残基之间，并同时与两条DNA链的可切割磷酸基团接触。一个与金属结合的水分子充当亲核试剂，是由酶侧链定位的广泛有序水分子网络的一部分。这些结构说明了高度多样化的LAGLIDADG酶家族采用了双金属内切酶机制的一种独特变体。
