Requirement of divalent cations for DNA cleavage is a general feature of type II restriction enzymes with the exception of few members of this group. A new type II restriction endonuclease has been partially purified from Lactococcus lactis KLDS4. The enzyme was denoted as LlaKI and showed to recognize and cleave the same site as FokI. The enzyme displayed a denatured molecular weight of 50 kDa and behaved as a dimer in solution as evidenced by the size exclusion chromatography. To investigate the role of divalent cations in DNA cleavage by LlaKI, digestion reactions were carried out at different Mg2+, Mn2+, and Ca2+ concentrations. Unlike most of type II restriction endonucleases, LlaKI did not require divalent metal ions to cleave DNA and is one of the few metal-independent restriction endonucleases found in bacteria. The enzyme showed near-maximal levels of activity in 10？mM Tris-HCl pH 7.9, 50？mM NaCl, 10？mM MgCl2, and 1？mM dithiothreitol at 30°C. The presence of DNA modification was also determined and was correlated with the correspondent restriction enzyme. 1. Introduction Restriction-modification enzymes are believed to function as a primitive bacterial “immune” system. They represent the main bacterial protection system against bacteriophage attacks. Restriction-modification systems are composed of two opposing activities: a methyltransferase that protects the host DNA against restriction by methylating the adenine or cytosine residues at certain recognition sites [1, 2] and a restriction endonuclease that recognizes and catalyzes double-strand cleavage of the same sequence if it is unmodified [3, 4]. A large number of restriction-modification systems have been discovered and well characterized during the past few decades; they occur ubiquitously among bacteria and their phages [5]. There are currently 3945 biochemically or genetically characterized restriction enzymes in REBASE and of the 3834 type II restriction enzymes, 299 distinct specificities are known [6]. Based on the cutting position, recognition sequence, cleavage requirements, and subunit structure, R-M systems are mainly classified into four types I, II, III, and IV. The type II R-M systems are the most abundant group of enzymes; they produce double-stranded DNA cleavage within or close the recognition sequence which consists of 4- to 8-defined nucleotides that can be symmetric, asymmetric, unique, or degenerated [7]. Most of type II restriction endonucleases show an absolute requirement for divalent metal ions to catalyze in a charge repulsive, polyanionic context the cleavage of
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