Abstract
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.
Highlights
Restriction-modification enzymes are believed to function as a primitive bacterial “immune” system
Lactococcus lactis KLDS4 was isolated from cow milk and identified by conventional phenotypic methods and 16S ribosomal RNA gene sequencing
Restriction assays on unmethylated lambda DNA showed the presence of a type II restriction endonuclease which was tentatively named LlaKI according to the suggested nomenclature rules [7]
Summary
Restriction-modification enzymes are believed to function as a primitive bacterial “immune” system. 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]. Only three restriction enzymes, BfiI, BmrI, and PabI have been shown to be EDTA-resistant and do not require Mg2+ or other divalent metal ions for catalytic activity [14,15,16]. These enzymes should cleave DNA by a different mechanism. This paper describes the purification and characterization of a new divalent ion-independent restriction-modification system from Lactococcus lactis KLDS4 and its biochemical and biophysical properties
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