Chandrashekaran Siddamadappa, Saravanan Matheshwaran, Radha Deshpande R, Nagaraja Valakunja
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012 and Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India.
J Biol Chem. 2004 Nov 26;279(48):49736-40. doi: 10.1074/jbc.M409483200. Epub 2004 Sep 16.
The characteristic feature of type II restriction endonucleases (REases) is their exquisite sequence specificity and obligate Mg(2+) requirement for catalysis. Efficient cleavage of DNA only in the presence of Ca(2+) ions, comparable with that of Mg(2+), is previously not described. Most intriguingly, KpnI REase exhibits Ca(2+)-dependent specific DNA cleavage. Moreover, the enzyme is highly promiscuous in its cleavage pattern on plasmid DNAs in the presence of Mn(2+) or Mg(2+), with the complete suppression of promiscuous activity in the presence of Ca(2+). KpnI methyltransferase does not exhibit promiscuous activity unlike its cognate REase. The REase binds to oligonucleotides containing canonical and mapped noncanonical sites with comparable affinities. However, the extent of cleavage is varied depending on the metal ion and the sequence. The ability of the enzyme to be promiscuous or specific may reflect an evolutionary design. Based on the results, we suggest that the enzyme KpnI represents an REase evolving to attain higher sequence specificity from an ancient nonspecific nuclease.
II型限制性核酸内切酶（REases）的特征在于其极高的序列特异性以及催化过程中对Mg(2+)的严格需求。此前尚未有过关于仅在Ca(2+)离子存在下DNA能被高效切割且效率与Mg(2+)相当的报道。最引人注目的是，KpnI REase表现出Ca(2+)依赖的特异性DNA切割。此外，在Mn(2+)或Mg(2+)存在时，该酶对质粒DNA的切割模式高度混杂，但在Ca(2+)存在时这种混杂活性会被完全抑制。与同源的REase不同，KpnI甲基转移酶不表现出混杂活性。该REase以相当的亲和力结合含有标准位点和已定位非标准位点的寡核苷酸。然而，切割程度会因金属离子和序列的不同而有所变化。酶的混杂或特异性能力可能反映了一种进化设计。基于这些结果，我们认为KpnI酶代表了一种从古老的非特异性核酸酶进化而来以获得更高序列特异性的REase。
