Dual Role for Zn2+ in Maintaining Structural Integrity and Inducing DNA Sequence Specificity in a Promiscuous Endonuclease

Abstract

We describe two uncommon roles for Zn2+ in enzyme KpnI restriction endonuclease (REase). Among all of the REases studied, KpnI REase is unique in its DNA binding and cleavage characteristics. The enzyme is a poor discriminator of DNA sequences, cleaving DNA in a promiscuous manner in the presence of Mg2+. Unlike most Type II REases, the active site of the enzyme comprises an HNH motif, which can accommodate Mg2+, Mn2+, or Ca2+. Among these metal ions, Mg2+ and Mn2+ induce promiscuous cleavage by the enzyme, whereas Ca2+-bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase. We describe two uncommon roles for Zn2+ in enzyme KpnI restriction endonuclease (REase). Among all of the REases studied, KpnI REase is unique in its DNA binding and cleavage characteristics. The enzyme is a poor discriminator of DNA sequences, cleaving DNA in a promiscuous manner in the presence of Mg2+. Unlike most Type II REases, the active site of the enzyme comprises an HNH motif, which can accommodate Mg2+, Mn2+, or Ca2+. Among these metal ions, Mg2+ and Mn2+ induce promiscuous cleavage by the enzyme, whereas Ca2+-bound enzyme exhibits site-specific cleavage. Examination of the sequence of the protein revealed the presence of a zinc finger CCCH motif rarely found in proteins of prokaryotic origin. The zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in an enzyme, suggesting the distinct origin of KpnI REase. A large number of proteins have bound zinc ions, which contribute to protein stability and/or catalytic functions more widely than any other transition metal ions (1Coleman J.E. Annu. Rev. Biochem. 1992; 61: 897-946Crossref PubMed Scopus (858) Google Scholar, 2Vallee B.L. Kim D.S. Biochemistry. 1990; 29: 5647-5659Crossref PubMed Scopus (1536) Google Scholar). A catalytic role for zinc was first shown in the case of carbonic anhydrase (3Keilin D. Kim T. Biochem. J. 1940; 34: 1163-1176Crossref PubMed Google Scholar), and its structural role was first proposed and demonstrated for the transcription factor TFIIIA (4Hanas J.S. Kim D.J. Bogenhagen D.F. Wu F.Y. Wu C.W. J. Biol. Chem. 1983; 258: 14120-14125Abstract Full Text PDF PubMed Google Scholar, 5Miller J. Kim A.D. Klug A. EMBO J. 1985; 4: 1609-1614Crossref PubMed Scopus (1705) Google Scholar). Since then, the roles for Zn2+ in numerous zinc-binding proteins have been identified and characterized. In many examples, the role of zinc ion is neither strictly structural nor catalytic, as in aminoacyl-tRNA synthetases, where zinc is involved in amino acid discrimination (6Sankaranarayanan R. Kim A.C. Rees B. Bovee M. Caillet J. Romby P. Francklyn C.S. Moras D. Nat. Struct. Biol. 2000; 7: 461-465Crossref PubMed Scopus (142) Google Scholar). Zinc binding motifs are structurally diverse and are present among proteins that perform a broad range of functions in various cellular processes. For instance, the motifs play a role in DNA recognition, transcription activation, protein folding and assembly, and protein-protein interactions (7Laity J.H. Kim B.M. Wright P.E. Curr. Opin. Struct. Biol. 2001; 11: 39-46Crossref PubMed Scopus (1096) Google Scholar). Zinc binding is observed in different groups of nucleases, I-PpoI, I-TevI, T4 endonuclease VII, DNA repair endonuclease IV, colicin E7, and S1 nuclease (8Flick K.E. Kim M.S. Monnat R.J. Jr. Stoddard B. L. Nature. 1998; 394: 96-101Crossref PubMed Scopus (195) Google Scholar, 9Van Roey P. Kim C.A. Fox K.M. Belfort M. Derbyshire V. EMBO J. 2001; 20: 3631-3637Crossref PubMed Scopus (62) Google Scholar, 10Hosfield D.J. Kim Y. Haas B.J. Cunningham R.P. Tainer J.A. Cell. 1999; 98: 397-408Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 11Ko T.P. Kim C.C. Ku W.Y. Chak K.F. Yuan H.S. Structure. 1999; 7: 91-102Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 12Gite S. Kim V. Eur. J. Biochem. 1992; 210: 437-441Crossref PubMed Scopus (18) Google Scholar). The binding of zinc is important for structural stability of I-PpoI, I-TevI, and T4 endonuclease VII and for catalysis in endonuclease IV and colicin E7. Bioinformatic analysis showed that McrA has a zinc binding fold, suggested to be needed for structural integrity (13Bujnicki J.M. Kim M. Rychlewski L. Mol. Microbiol. 2000; 37: 1280-1281Crossref PubMed Scopus (22) Google Scholar). R.BslI contains two glucocorticoid receptor-like zinc (Cys4) binding motifs, which are important for the protein-DNA and protein-protein interactions (14Vanamee E.S. Kim P. Zhu Z. Yates D. Garman E. Xu S. Aggarwal A.K. J. Mol. Biol. 2003; 334: 595-603Crossref PubMed Scopus (8) Google Scholar). In this paper, we describe two distinct roles for Zn2+ in R.KpnI. Type II REases 3The abbreviations used are: REase, restriction endonuclease; WT, wild type. require Mg2+ or a similar divalent metal ion to cleave DNA. Almost 3700 Type II restriction enzymes, representing more than 262 distinct specificities, are known to date (15Roberts R.J. Kim T. Posfai J. Macelis D. Nucleic Acids Res. 2007; 35: D269-D270Crossref PubMed Scopus (207) Google Scholar). Most Type II REases belong to the PD... (D/E)XK superfamily (16Pingoud A. Kim M. Pingoud V. Wende W. Cell Mol. Life Sci. 2005; 62: 685-707Crossref PubMed Scopus (376) Google Scholar). Recent structural and bioinformatics studies revealed that apart from the PD... (D/E)XK superfamily, few REases belong to other nuclease superfamilies, such as Nuc, HNH, and YIG-GIY, which are structurally unrelated to each other (17Sapranauskas R. Kim G. Lagunavicius A. Vilkaitis G. Lubys A. Siksnys V. J. Biol. Chem. 2000; 275: 30878-30885Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 18Aravind L. Kim K.S. Koonin E.V. Nucleic Acids Res. 2000; 28: 3417-3432Crossref PubMed Google Scholar, 19Bujnicki J.M. Kim M. Rychlewski L. Trends Biochem. Sci. 2001; 26: 9-11Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Sequence alignment and subsequent validation experiments showed that R.KpnI is the first member of the HNH superfamily (20Saravanan M. Kim J.M. Cymerman I.A. Rao D.N. Nagaraja V. Nucleic Acids Res. 2004; 32: 6129-6135Crossref PubMed Scopus (70) Google Scholar). Although at first glance R.KpnI appeared to be a typical dimeric Type IIP REase recognizing and cleaving palindromic sequence GGTACC, it has several distinct features. The properties include prolific promiscuous activity in the presence of Mg2+ which is further enhanced with Mn2+, efficient site specific high fidelity DNA cleavage when Ca2+ is used instead of Mg2+, and suppression of the promiscuous cleavage activity in presence of Ca2+ (21Chandrashekaran S. Kim M. Radha D.R. Nagaraja V. J. Biol. Chem. 2004; 279: 49736-49740Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Kinetic studies revealed that the Ca2+-mediated exquisite specificity is achieved at the step of DNA cleavage (22Saravanan M. Kim K. Kanakaraj R. Rao D.N. Nagaraja Nucleic Acids Res. 2007; 35: 2777-2786Crossref PubMed Scopus (22) Google Scholar). The alignment of McrA, T4 endonuclease VII, and R.KpnI is depicted in Fig. 1A. The former two enzymes have tetra-Cys Zn2+ fingers, whereas R.KpnI has an unusual CCCH putative Zn2+ finger. Here we describe the importance of the Zn2+ finger motif in Zn2+ coordination. Surprisingly, the bound Zn2+ has more complex, multiple roles in R.KpnI function, in a manner distinct from any other restriction-modification system. Enzymes and DNA—T4 polynucleotide kinase, Pfu DNA polymerase, and DpnI were purchased from New England Biolabs. Oligonucleotides (Sigma and Microsynth) were purified on 18% urea-polyacrylamide gel (33Sambrook J. Kim E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and end-labeled with T4 polynucleotide kinase and [γ-32P]ATP (6000 Ci/mmol). Mutagenesis, Expression, and Purification of Mutant Proteins—The model of R.KpnI was built using the structure of T4 endonuclease VII and other structurally characterized HNH superfamily nucleases. The detailed procedure has been described and discussed previously (20Saravanan M. Kim J.M. Cymerman I.A. Rao D.N. Nagaraja V. Nucleic Acids Res. 2004; 32: 6129-6135Crossref PubMed Scopus (70) Google Scholar). Site-directed mutagenesis was performed by the megaprimer method (23Kirsch R.D. Kim E. Nucleic Acids Res. 1998; 26: 1848-1850Crossref PubMed Scopus (206) Google Scholar). The mutations were confirmed by sequencing. The WT and mutants were expressed in Escherichia coli BL26 (F-omp T hsdSB (rB- mB-) gal dcm Δlac (DE3) nin5 lac UV5-T7 gene 1) containing KpnI methyltransferase, and the cells were induced with 0.3 mm isopropyl-β-d-thiogalactopyranoside as described previously (24Chandrashekaran S. Kim P. Nagaraja V. J. Biosci. 1999; 24: 269-277Crossref Scopus (14) Google Scholar). Cells were lysed by sonication in buffer A containing 10 mm potassium phosphate (pH 7.0), 1 mm EDTA, 7 mm 2-mercaptoethanol. The supernatant was subjected to 0-50% ammonium sulfate fractionation. The samples were dialyzed against buffer A and purified by phosphocellulose and Hi-Trap heparin columns. The fractions containing the enzyme were pooled and dialyzed against buffer B (10 mm Tris-HCl (pH 7.4), 0.1 mm EDTA, 50 mm KCl, 5 mm 2-mercaptoethanol, and 50% glycerol). The concentration of the proteins was estimated by the method of Bradford (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). Atomic Absorption Analysis—Purified R.KpnI (10 mg) was denatured and renatured in the presence or absence of 100 μm ZnCl2 or 5 mm EDTA and then dialyzed overnight against 20 mm Tris-HCl (pH 7.4), 150 mm NaCl at 4 °C with buffer changes to eliminate excess metal ions or chelators. Chelex-100 resin (Sigma) was used to remove trace metal ions in all of the buffers. The samples were analyzed by atomic absorption spectroscopy. The dialyzed buffer after Chelex treatment was used as a blank, and the residual Zn2+ background was subtracted from the measurement of protein samples. Zn2+ Blotting Assay—Purified R.KpnI and its mutants (0-6 μg) was slot-blotted onto nitrocellulose membrane presoaked in buffer (10 mm Tris, pH 7.5, 100 mm NaCl, 1 mm EDTA). Proper transfer was ascertained by Ponceau-S staining with transferred protein amounts estimated using Quantity One software. After transfer, the membrane was incubated at 37 °C for 1 h in buffer containing 10 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10 mm MgCl2 and subsequently washed three times (15 min each) in the same buffer. The membrane was next incubated in buffer containing 30 μCi of 65ZnCl2 (specific activity, 800 mCi/g; BARC, Mumbai) at room temperature for 1 h with gentle rocking. The unbound radioactivity was removed by washing the membrane three times with buffer (20 min each). The membrane was dried and exposed to a PhosphorImager screen. Electrophoretic Mobility Shift Assay—Different concentrations of the WT (1-10 nm) and mutant R.KpnI (10-100 nm) were incubated with the 0.2 pmol of end-labeled double-stranded oligonucleotide (20-mer) containing the R.KpnI recognition site in binding buffer (20 mm Tris-HCl (pH 7.4), 25 mm NaCl, and 5 mm 2-mercaptoethanol) for 15 min on ice. The free DNA and the enzyme-bound complexes were resolved by 8% native polyacrylamide gel electrophoresis in 1× TBE buffer (89 mm Tris-HCl, 89 mm boric acid, and 1 mm EDTA), and then signals were detected by autoradiography. In Vitro DNA Cleavage and Steady-state Kinetic Analysis—Purified R.KpnI and its mutants were incubated with 500 ng of plasmid DNA in buffer containing 10 mm Tris-HCl (pH 7.4), 5 mm 2-mercaptoethanol, 2 mm MgCl2, or 10-100 μm ZnCl2 for 1 h at 37 °C. The cleavage products were analyzed on 1% agarose gel. For kinetic analysis, the purified enzyme was dialyzed against 10 mm EDTA to remove any bound metal ions. Steady-state kinetic time courses with canonical DNA substrates were measured at DNA concentrations of 5-150-fold molar excess over dimeric enzyme (1 nm) in the presence of 100 μm Zn2+. The kinetic parameters were determined as described (22Saravanan M. Kim K. Kanakaraj R. Rao D.N. Nagaraja Nucleic Acids Res. 2007; 35: 2777-2786Crossref PubMed Scopus (22) Google Scholar). Circular wild R.KpnI and its mutants the and mutations were analyzed by The were at 25 °C from to using a and a of 0.2 The were at of 50 and were of the samples were The buffer used was 50 mm Tris-HCl (pH 7.4), mm NaCl, 1 mm 2-mercaptoethanol. The proteins were at a concentration of 0.2 and the molar was using the where is the observed is is concentration is the of the in to the number of and is stability of the protein samples was using by changes in the with temperature A nm) was to the protein and the at that is as the temperature is of of different samples of R.KpnI and its mutants (1 were in 50 mm Tris-HCl pH and at 37 °C using 1% samples μg) were after various time and was with buffer containing were analyzed by R.KpnI Zn2+ analysis and of R.KpnI the presence of an unusual CCCH zinc finger motif (20Saravanan M. Kim J.M. Cymerman I.A. Rao D.N. Nagaraja V. Nucleic Acids Res. 2004; 32: 6129-6135Crossref PubMed Scopus (70) Google Scholar) different from other previously described found zinc finger motifs The putative are shown in the model The of and in R.KpnI is among zinc finger proteins of prokaryotic origin. the bound atomic absorption dialyzed R.KpnI was found to 2 of of The dimeric of the enzyme has been (24Chandrashekaran S. Kim P. Nagaraja V. J. Biosci. 1999; 24: 269-277Crossref Scopus (14) Google Scholar). Zn2+ was with other metal ions, after against a buffer that 10 mm 2 of Zn2+ were in the that the site was to by Mg2+. when protein was renatured in presence of the zinc to 4 of R.KpnI of this in the presence of MgCl2 in of 2 of Zn2+ from the of two of the Zn2+ ions by Mg2+. The other 2 of tightly bound zinc be by Mg2+. that R.KpnI two zinc-binding is with Mg2+, and another is atomic absorption of μm mm mm in a of the Zn2+ in the role of zinc in we the DNA binding and cleavage properties of in the absence of and The R.KpnI with Zn2+ DNA The enzyme DNA cleavage activity in the presence of 2 mm experiments were with native and zinc R.KpnI The enzyme binds and the DNA in a promiscuous manner in similar to the native enzyme. the of DNA binding and cleavage in R.KpnI is to the structural in the we In presence of the of R.KpnI has two at and which is a of The enzyme showed structure with enzyme, the importance of Zn2+ to the structure of the R.KpnI The stability of these proteins was by were measured at from 30 to °C. The of enzyme was by °C over the enzyme, a role for Zn2+ in stability of the enzyme In with and experiments showed that the enzyme is more to cleavage than the native or enzyme We that Zn2+ is for of the active R.KpnI in R.KpnI. of R.KpnI. are the of for and R.KpnI in structure were by from to nm) for and R.KpnI the of three using protein concentrations of of and R.KpnI. The detailed procedure is described The were resolved on The CCCH in Zn2+ and of the R.KpnI that the zinc binding is the unusual zinc finger motif shown in Fig. mutations were in R.KpnI. The and and of the putative motif were by The mutant proteins were analyzed for zinc binding using a zinc of the mutants to zinc in to R.KpnI. Zn2+ has been shown to be for the folding and stability of many Zn2+ finger Zinc experiments that and are for Zn2+ in R.KpnI. We the of in Zn2+ on the stability of the enzyme by the of the mutant The at as a of temperature for WT and mutants is shown in Fig. The mutant proteins showed stability with that of the WT that the mutations at the CCCH motif the folding of the enzyme. the mutant enzymes showed with the importance of the CCCH motif for the structural stability of the enzyme of CCCH Zn2+ on DNA Cleavage and and its mutants were analyzed for the to cleave DNA. the DNA was by 1 WT enzyme, was cleavage observed with all the mutants at a excess of the enzyme the for the of DNA cleavage observed with the we analyzed the DNA binding of the mutants by The mutants to the DNA containing R.KpnI recognition sequence The mutants showed DNA binding and to the of the structure as observed in and that the of with zinc the structural integrity of the the activity of the enzyme. The of DNA binding and cleavage with amino acid in R.KpnI is a typical of Zn2+ finger proteins where Zn2+ has a structural DNA Cleavage and of Mg2+- and atomic absorption analysis of R.KpnI showed that the enzyme binds 4 of zinc Among the 4 2 can be with Mg2+. This at the of zinc ions binding to the active site to the properties of R.KpnI in addition to the at the CCCH The 2 of Zn2+ bound the Mg2+ DNA cleavage. Surprisingly, the enzyme showed efficient DNA cleavage in the presence of 50 μm Zn2+ the role of Zn2+ in the specificity of we DNA cleavage experiments at enzyme concentrations nm) and in the presence of 100 μm Zn2+. at such high concentrations of the enzyme, promiscuous cleavage is in In experiments using of the as a in the presence of 2 mm Mg2+ or 100 μm the cleavage was observed with Mg2+, that the enzyme is specific in the presence of Zn2+ DNA cleavage was observed in the presence of Zn2+ with any of the other In the experiments described enzyme activity appeared to be with the activity in the presence of other metal ions. We to kinetic analysis to Zn2+ DNA cleavage. Kinetic analysis in the presence of Zn2+ revealed the number of the enzyme to be which is with that of Ca2+ that DNA cleavage is as efficient as cleavage (22Saravanan M. Kim K. Kanakaraj R. Rao D.N. Nagaraja Nucleic Acids Res. 2007; 35: 2777-2786Crossref PubMed Scopus (22) Google Scholar). The of the enzyme for the canonical sequence with Zn2+ nm) is similar to that of Mg2+ The of Zn2+ to Mg2+ from the active site and induce specific cleavage that it suppress the promiscuous activity of the enzyme. of Zn2+ experiments on Mg2+- or showed that the Mg2+- or Mn2+-mediated promiscuous activity was in the presence of 100 μm Zn2+ The of different metal ions to the active site the of the active studies revealed that R.KpnI the HNH motif in its for DNA cleavage. and the active site and are for Mg2+ binding and catalysis (20Saravanan M. Kim J.M. Cymerman I.A. Rao D.N. Nagaraja V. Nucleic Acids Res. 2004; 32: 6129-6135Crossref PubMed Scopus (70) Google Scholar). The active site mutant enzymes and were analyzed for DNA cleavage Fig. cleavage on the same catalytic motif, the of the R.KpnI active site HNH motif to accommodate Mg2+, Mn2+, and Zn2+. of the other metal ions from the active site by Zn2+ and of the catalytic activity with kinetic with of Mg2+, Mn2+, and Ca2+ that Zn2+ is a for parameters of with different divalent metal from 0.3 from from 0.1 Kinetic from (22Saravanan M. Kim K. Kanakaraj R. Rao D.N. Nagaraja Nucleic Acids Res. 2007; 35: 2777-2786Crossref PubMed Scopus (22) Google Scholar). in a Zinc finger proteins are involved in cellular and (7Laity J.H. Kim B.M. Wright P.E. Curr. Opin. Struct. Biol. 2001; 11: 39-46Crossref PubMed Scopus (1096) Google Scholar). Zinc finger motifs have been and in of the structural in a number of Roey P. Kim C.A. Fox K.M. Belfort M. Derbyshire V. EMBO J. 2001; 20: 3631-3637Crossref PubMed Scopus (62) Google Scholar, E.S. Kim P. Zhu Z. Yates D. Garman E. Xu S. Aggarwal A.K. J. Mol. Biol. 2003; 334: 595-603Crossref PubMed Scopus (8) Google Scholar, Kim S. B. D. EMBO J. 1999; PubMed Scopus Google Scholar). We that a sequence motif found in R.KpnI is a zinc binding on the of and/or several of zinc finger and have been characterized and shown to be involved in interactions with or other proteins J.M. Kim Annu. Rev. Struct. 26: PubMed Scopus Google Scholar). zinc were identified in a number of proteins of origin Kim J.M. Biochemistry. 2003; PubMed Scopus Google Scholar) and found in which is for the of active Kim G. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The of three and a in the CCCH zinc finger found in R.KpnI is in prokaryotic The CCCH zinc were identified in protein A in different of Y. Kim D. J. 2005; PubMed Scopus (14) Google Scholar), from K. Kim Y. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) and 50 protein from Kim J. Res. 2001; PubMed Scopus Google Scholar). R.KpnI is a member of the CCCH Zn2+ finger and is the first REase to have this The Zn2+ in other this motif have roles in protein-protein acid structural and the in Fig. it is that tightly bound Zn2+ has a structural it promiscuous cleavage. The Zn2+ which is bound to the active catalytic function. A of R.KpnI is its promiscuous behavior in the presence of Mg2+ with any other REase (21Chandrashekaran S. Kim M. Radha D.R. Nagaraja V. J. Biol. Chem. 2004; 279: 49736-49740Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The Mg2+ promiscuous cleavage by R.KpnI is by Zn2+ the high fidelity cleavage. The of the R.KpnI active site the binding of Mg2+, Mn2+, or which have different and to induce promiscuous or specific cleavage. in Zn2+ has structural and catalytic found in any enzyme The tightly bound Zn2+ at the CCCH motif structural integrity for the enzyme, whereas the Zn2+ at the active site high specificity cleavage. Zn2+ finger motifs as such to be in of prokaryotic origin. Although the HNH motif is found in diverse of nucleases, the zinc finger motifs are found in McrA, I-PpoI, and T4 endonuclease VII to the Although the zinc finger of T4 endonuclease VII has a structural the of similar zinc finger in McrA is The two Zn2+ and in are important for structural of the protein The catalytic and structural role for Zn2+ in R.KpnI at its origin and in We J. M. for the R.KpnI and the of and Molecular and for and atomic absorption with