Lykke-Andersen J, Garrett R A, Kjems J
Institute of Molecular Biology, Copenhagen University, Denmark.
Nucleic Acids Res. 1996 Oct 15;24(20):3982-9. doi: 10.1093/nar/24.20.3982.
The archaeal intron-encoded homing enzymes I-PorI and I-DmoI belong to a family of endonucleases that contain two copies of a characteristic LAGLIDADG motif. These endonucleases cleave their intron- or intein- alleles site-specifically, and thereby facilitate homing of the introns or inteins which encode them. The protein structure and the mechanism of DNA recognition of these homing enzymes is largely unknown. Therefore, we examined these properties of I-PorI and I-DmoI by protein footprinting. Both proteins were susceptible to proteolytic cleavage within regions that are equidistant from each of the two LAGLIDADG motifs. When complexed with their DNA substrates, a characteristic subset of the exposed sites, located in regions immediately after and 40-60 amino acids after each of the LAGLIDADG motifs, were protected. Our data suggest that the enzymes are structured into two, tandemly repeated, domains, each containing both the LAGLIDADG motif and two putative DNA binding regions. The latter contains a potentially novel DNA binding motif conserved in archaeal homing enzymes. The results are consistent with a model where the LAGLIDADG endonucleases bind to their non-palindromic substrates as monomeric enzymes, with each of the two domains recognizing one half of the DNA substrate.
古菌内含子编码的归巢酶I-PorI和I-DmoI属于一类核酸内切酶家族，这类酶含有两个特征性LAGLIDADG基序拷贝。这些核酸内切酶能位点特异性地切割它们的内含子或内含肽等位基因，从而促进编码它们的内含子或内含肽的归巢。这些归巢酶的蛋白质结构和DNA识别机制在很大程度上尚不清楚。因此，我们通过蛋白质足迹法研究了I-PorI和I-DmoI的这些特性。两种蛋白质在与两个LAGLIDADG基序中每个基序距离相等的区域内都易于被蛋白酶切割。当与它们的DNA底物结合时，位于每个LAGLIDADG基序之后紧邻区域以及之后40-60个氨基酸处的暴露位点的一个特征性子集受到保护。我们的数据表明，这些酶被构建成两个串联重复的结构域，每个结构域都包含LAGLIDADG基序和两个假定的DNA结合区域。后者包含一个在古菌归巢酶中保守的潜在新型DNA结合基序。这些结果与一个模型一致，即LAGLIDADG核酸内切酶作为单体酶与其非回文底物结合，两个结构域中的每个结构域识别DNA底物的一半。
