%0 Journal Article
%T A Sequence-Specific Nicking Endonuclease from Streptomyces: Purification, Physical and Catalytic Properties
%A Peechapack Somyoonsap
%A Vichein Kitpreechavanich
%A Somchai Pornbanlualap
%J ISRN Biochemistry
%D 2013
%R 10.1155/2013/287158
%X A sequence-specific nicking endonuclease from Streptomyces designated as DC13 was purified to near homogeneity. Starting with 30 grams of wet cells, the enzyme was purified by ammonium sulfate fractionation, DEAE cellulose, and phenyl-Sepharose chromatography. The purified protein had a specific activity 1000 units/mg and migrated on SDS-PAGE gel with an estimated molecular weight of 71£¿kDa. Determination of subunit composition by gel filtration chromatography indicated that the native enzyme is a monomer. When incubated with different DNA substrates including pBluescript II KS, pUC118, pET-15b, and pET-26b, the enzyme converted these supercoiled plasmids to a mixture of open circular and linear DNA products, with the open circular DNA as the major cleavage product. Analysis of the kinetic of DNA cleavage showed that the enzyme appeared to cleave super-coiled plasmid in two distinct steps: a rapid cleavage of super-coiled plasmid to an open circular DNA followed a much slower step to linear DNA. The DNA cleavage reaction of the enzyme required Mg2+ as a cofactor. Based on the monomeric nature of the enzyme, the kinetics of DNA cleavage exhibited by the enzyme, and cofactor requirement, it is suggested here that the purified enzyme is a sequence-specific nicking endonuclease that is similar to type IIS restriction endonuclease. 1. Introduction Restriction enzymes recognizing unique DNA sequences are indispensable tools in molecular biology. Among three different types of restriction enzymes that have been described, type II endonucleases are the most useful enzymes for gene cloning because they recognize 4¨C8£¿bp sequences and cleave the DNA within or in close proximity to the recognition site [1]. More than 3500 different type II restriction endonucleases with 240 unique specificities have been currently identified [2]. Restriction endonucleases working with methyltransferases form the basis of RE (restriction modification) systems of prokaryotes to protect them against the invasion of foreign DNA [3]. Type II endonucleases consist of structurally and mechanistically diverse subclasses of enzymes, each with a different mechanism of sequence recognition, DNA cleavage, and organization of quaternary structure [4]. The orthodox type IIP enzymes are the most studied and include the majority of enzymes such as EcoRI, EcoRV, and BamHI commonly used in molecular biology [5]. These enzymes typically exist in solution as a homodimer, bind to a symmetrical palindromic 4¨C8£¿bp sequence as a dimer, and cleave phosphodiester bonds on both strands of DNA within the
%U http://www.hindawi.com/journals/isrn.biochemistry/2013/287158/