Cymerman Iwona A, Obarska Agnieszka, Skowronek Krzysztof J, Lubys Arvydas, Bujnicki Janusz M
Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
Proteins. 2006 Dec 1;65(4):867-76. doi: 10.1002/prot.21156.
The restriction endonuclease (REase) R. HphI is a Type IIS enzyme that recognizes the asymmetric target DNA sequence 5'-GGTGA-3' and in the presence of Mg(2+) hydrolyzes phosphodiester bonds in both strands of the DNA at a distance of 8 nucleotides towards the 3' side of the target, producing a 1 nucleotide 3'-staggered cut in an unspecified sequence at this position. REases are typically ORFans that exhibit little similarity to each other and to any proteins in the database. However, bioinformatics analyses revealed that R.HphI is a member of a relatively big sequence family with a conserved C-terminal domain and a variable N-terminal domain. We predict that the C-terminal domains of proteins from this family correspond to the nuclease domain of the HNH superfamily rather than to the most common PD-(D/E)XK superfamily of nucleases. We constructed a three-dimensional model of the R.HphI catalytic domain and validated our predictions by site-directed mutagenesis and studies of DNA-binding and catalytic activities of the mutant proteins. We also analyzed the genomic neighborhood of R.HphI homologs and found that putative nucleases accompanied by a DNA methyltransferase (i.e. predicted REases) do not form a single group on a phylogenetic tree, but are dispersed among free-standing putative nucleases. This suggests that nucleases from the HNH superfamily were independently recruited to become REases in the context of RM systems multiple times in the evolution and that members of the HNH superfamily may be much more frequent among the so far unassigned REase sequences than previously thought.
限制性内切核酸酶（REase）R. HphI是一种IIS型酶，它识别不对称靶DNA序列5'-GGTGA-3'，并在Mg(2+)存在的情况下，在靶序列3'侧8个核苷酸处水解DNA两条链中的磷酸二酯键，在该位置的未指定序列中产生1个核苷酸的3'端交错切割。REase通常是孤儿蛋白，彼此之间以及与数据库中的任何蛋白质都几乎没有相似性。然而，生物信息学分析表明，R.HphI是一个相对较大的序列家族的成员，该家族具有保守的C端结构域和可变的N端结构域。我们预测，该家族蛋白质的C端结构域对应于HNH超家族的核酸酶结构域，而不是最常见的PD-(D/E)XK核酸酶超家族。我们构建了R.HphI催化结构域的三维模型，并通过定点诱变以及对突变蛋白的DNA结合和催化活性的研究验证了我们的预测。我们还分析了R.HphI同源物的基因组邻域，发现伴有DNA甲基转移酶的推定核酸酶（即预测的REase）在系统发育树上并不形成一个单一的群体，而是分散在独立的推定核酸酶之中。这表明，在进化过程中，HNH超家族的核酸酶多次在RM系统的背景下被独立招募成为REase，并且HNH超家族的成员在迄今为止未分类的REase序列中可能比以前认为的更为常见。
