Ca2+-mediated Site-specific DNA Cleavage and Suppression of Promiscuous Activity of KpnI Restriction Endonuclease

Abstract

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.Type II REases recognize specific DNA sequences, which vary from 4 to 8 bp and cleave both the strands in Mg 2ϩ -dependent reaction. They exhibit remarkable features in discriminating between specific and nonspecific DNA, not only in DNA binding but also during DNA cleavage (1). Based on the divalent metal ion requirement for DNA binding, REases have been classified into two groups (2). For example, EcoRI, BamHI, RsrI, and a few other enzymes bind to DNA preferentially at their recognition sites in the absence of divalent metal ions (3-5). In contrast, EcoRV, PvuII, TaqI, and a few other enzymes bind to cognate sequences in a divalent metal ion-dependent manner (6 -8). However, both the groups of enzymes need Mg 2ϩ for catalysis. KpnI REase isolated from Klebsiella pneumoniae recognizes the palindromic double-stranded DNA sequence 5Ј-GGTAC2C-3Ј and cleaves DNA by generating a 3Ј, 4-base overhang (18). The enzyme binds to specific DNA in the absence of divalent metal ions (19). In this paper, we describe the highly promiscuous cleavage of DNA in the presence of Mg 2ϩ and Mn 2ϩ . The promiscuous activity is completely suppressed in the presence of Ca 2ϩ , which induces site-specific DNA cleavage. EXPERIMENTAL PROCEDURESEnzymes and DNA-KpnI REase was purified as described previously (18). The enzyme was diluted in binding buffer (20 mM Tris-HCl (pH 7.4), 25 mM NaCl, and 5 mM 2-mercaptoethanol) for all the studies. The concentration of the enzyme was estimated by the method of Bradford (20). One unit of KpnI REase is defined as the amount of enzyme required for complete digestion of 1 g of DNA at 37°C for 1 h by using assay buffer containing 5 mM Mg 2ϩ . T4 polynucleotide kinase and Klenow polymerase were purchased from New England Biolabs. Oligonucleotides were from Microsynth Inc., Switzerland, and purified on 18% urea-polyacrylamide gel (21). The purified oligonucleotides were end-labeled with T4 polynucleotide kinase and [␥-32 P]ATP ...

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

“…However, a comparison of the cofactor preferences of PD-(D/E)XK and HNH REases indicated that HNH enzymes have a greater flexibility for metal ion coordination. For example, R.KpnI, the first REase member of the HNH superfamily to be identified, exhibits broad cofactor utilization for DNA cleavage (10,149,150). This utilization is also seen in nonspecific endonucleases belonging to the superfamily, e.g., colicin E7, colicin E9, and Serratia nuclease (151)(152)(153).…”

Promiscuous restriction is a cellular defense strategy that confers fitness advantage to bacteria

“…Modification of the phage genome was carried out by preparing the phage lysates on E. coli BL26 strain overexpressing KpnI MTase (pUCMK). M.KpnI does not exhibit promiscuous behavior and methylates in a site-specific manner (14). The modification status of the phage genomes was confirmed by resistance to site-specific cleavage by KpnI REase both in vitro and in vivo.…”

“…The dsDNA breaks catalyzed by REases could be lethal to the host genome if they are not controlled (26). The host cell harboring promiscuous R.KpnI and site-specific M.KpnI (14) needs to protect its genome not only at the canonical sites but at the noncanonical sites. In case of type I R-M systems, it is established that the resident chromosome is recognized as self rather than foreign even in the absence of modification, a phenomenon known as "restriction alleviation" (27,28).…”

“…Studies carried out with R.KpnI, a type II REase, opened up a different perspective on this prevailing theme, because the enzyme exhibits highly promiscuous cleavage under certain conditions (14). The REase exhibits remarkable versatility in substrate recognition and cofactor utilization, whereas the cognate MTase is very site-specific (14).…”

“…The REase exhibits remarkable versatility in substrate recognition and cofactor utilization, whereas the cognate MTase is very site-specific (14). In addition to its recognition sequence, GGTACC, the enzyme binds and cleaves a number of noncanonical sequences (i.e., TGTACC, GTTACC, GATACC, GGAACC, GGTCCC, GGTATC, GGTACG, GGTACT), with the binding affinity ranging from 8 to 35 nM (14,15). Accordingly, many of these sites are cleaved efficiently with k cat values varying from 0.06 to 0.15 min −1 vs. 4.3 min −1 for canonical sites (15).…”