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A Jeltsch

Publications and source records attributed to A Jeltsch.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mutational analysis of target base flipping by the EcoRV adenine-N6 DNA methyltransferase.

DNA methyltransferases flip their target base out of the DNA helix. Here, we have investigated base flipping by wild-type EcoRV DNA methyltransferase (M.EcoRV) and five M.EcoRV variants (D193A, Y196A, S229A, W231R and Y258A). These variants bind to DNA and S-adenosylmethionine but have a severely reduced catalytic efficiency or are catalytically inactive. To measure base flipping three different assays were used, viz. analysis of the yields of photocrosslinking reactions between the enzymes and a substrate in which the target base is replaced by 5-iodouracil, analysis of the binding constants to substrates containing a mismatch base-pair at the target position and analysis of the salt dependence of specific complex formation. Our data show that the Y196A, W231R and Y258A variants are not able to stabilize a flipped target base, suggesting that the aromatic amino acid residues (Tyr196, Trp231 and Tyr258) are involved in hydrophobic interactions with the flipped base. The D193A variant behaves like wild-type M.EcoRV with respect to base flipping. The fact that this variant is catalytically inactive indicates that Asp193 has a function in chemical catalysis. The S229A variant can better flip modified bases but does not tightly lock the flipped base into the adenine-binding pocket, suggesting that Ser229 could form a contact to the flipped adenine.

Cross-Linking Reagents↗
PubMed1999

Circular permutations in the molecular evolution of DNA methyltransferases.

Circular permutations of genes during molecular evolution often are regarded as elusive, although a simple model can explain these rearrangements. The model assumes that first a gene duplication of the precursor gene occurs in such a way that both genes become fused in frame, leading to a tandem protein. After generation of a new start codon within the 5' part of the tandem gene and a stop at an equivalent position in the 3' part of the gene, a protein is encoded that represents a perfect circular permutation of the precursor gene product. The model is illustrated here by the molecular evolution of adenine-N6 DNA methyltransferases. beta- and gamma-type enzymes of this family can be interconverted by a single circular permutation event. Interestingly, tandem proteins, proposed as evolutionary intermediates during circular permutation, can be directly observed in the case of adenine methyltransferases, because some enzymes belonging to type IIS, like the FokI methyltransferase, are built up by two fused enzymes, both of which are active independently of each other. The mechanism for circular permutation illustrated here is very easy and applicable to every protein. Thus, circular permutation can be regarded as a normal process in molecular evolution and a changed order of conserved amino acid motifs should not be interpreted to argue against divergent evolution.

DNA Modification Methylases↗
PubMed1998

Crosslinking the EcoRV restriction endonuclease across the DNA-binding site reveals transient intermediates and conformational changes of the enzyme during DNA binding and catalytic turnover.

EcoRV completely encircles bound DNA with two loops, forming the entry and exit gate for the DNA substrate. These loops were crosslinked generating CL-EcoRV which binds and releases linear DNA only slowly, because threading linear DNA into and out of the DNA-binding 'tunnel' of CL-EcoRV is not very effective. If the crosslinking reaction is carried out with a circular bound DNA, CL-EcoRV is hyperactive towards the trapped substrate which is cleaved very quickly but not very accurately. CL-EcoRV also binds to, but does not cleave, circular DNA when added from the outside, because it cannot enter the active site. Based on these results a two-step binding model is proposed for EcoRV: initial DNA binding occurs at the outer side of the loops before the gate opens and then the DNA is transferred to the catalytic center.

Amino Acid Substitution↗
PubMed1998

Protein engineering of the restriction endonuclease EcoRV--structure-guided design of enzyme variants that recognize the base pairs flanking the recognition site.

We generated variants of the restriction endonuclease EcoRV that discriminate between recognition sites with different flanking sequences. This was achieved by designing new contacts to the bases in the major groove of the DNA preceding and following the EcoRV recognition site. We selected Ala181 as the starting point for the extension of the site specificity of EcoRV because, according to the structure of the specific EcoRV x DNA complex, this residue is involved in a water mediated contact with the bases flanking the recognition sequence on the 5' side. A substitution of this alanine residue by other amino acid residues changes the protein-DNA interface in this region and potentially creates new contacts, such that EcoRV variants could have an extended specificity, i.e. a greater selectivity for EcoRV sites within a particular sequence context. EcoRV variants with naturally occurring amino acid residues at position 181 were produced and their selectivity analyzed with oligodeoxynucleotide and plasmid substrates that differ only in the base pairs immediately flanking the EcoRV site. Some variants, having amino acid residues with long or bulky side chains at position 181 showed altered preferences for the base pairs flanking the recognition sequence with oligodeoxynucleotide substrates without loosing their catalytic efficiency. One variant, A181K, is able to discriminate between purine and pyrimidine bases on the 5' side of the recognition sequence, probably by means of a new hydrogen bond to the N7 of the purine base. Another variant, A181E, strongly prefers a thymine base on the 5' side of the recognition sequence, presumably due to a hydrogen bond formed between the protonated glutamic acid residue and the O4 of thymine.

Base Sequence↗
PubMed1998

Flexibility of DNA in complex with proteins deduced from the distribution of bending angles observed by scanning force microscopy.

Flexibility and dynamics of DNA are important for DNA-binding and recognition by proteins. Here the flexibility of DNA is calculated from the distribution of DNA-bending angles of single DNA molecules as observed by scanning force microscopy by applying an equation that links the force constant of DNA-bending (f) to the variance of the distribution of bending angles (sigma): f=RT/sigma(2). Using published data, f is calculated to be 3-5 J/degree(2) for free DNA. Thus, bending DNA by 20 degrees requires approx. 0.5-1 kJ/mol. This result shows that DNA is very flexible and readily can be bent by thermal motion. DNA-flexibility is not altered in some protein-DNA complexes (HhaI methyltransferase, EcoRV restriction endonuclease). In contrast, DNA-binding by EcoRI endonuclease increases DNA-flexibility and binding by EcoRI methyltransferase restricts the flexibility of DNA. During the transition of the RNA polymerase-sigma(54)-DNA complex from the closed to the open form and of cro repressor from a non-specific to a specific binding mode the flexibility of the DNA is strongly reduced.

Journal Article↗
PubMed1998

Functional roles of conserved amino acid residues in DNA methyltransferases investigated by site-directed mutagenesis of the EcoRV adenine-N6-methyltransferase.

All DNA methyltransferases (MTases) have similar catalytic domains containing nine blocks of conserved amino acid residues. We have investigated by site-directed mutagenesis the function of 17 conserved residues in the EcoRV alpha-adenine-N6-DNA methyltransferase. The structure of this class of MTases has been predicted recently. The variants were characterized with respect to their catalytic activities and their abilities to bind to DNA and the S-adenosylmethionine (AdoMet) cofactor. Amino acids located in motifs X, I, and II are shown to be involved in AdoMet binding (Lys16, Glu37, Phe39, and Asp58). Some of the mutants defective in AdoMet binding are also impaired in DNA binding, suggesting allosteric interactions between the AdoMet and DNA binding site. Asp78 (motif III), which was supposed to form a hydrogen bond to the AdoMet on the basis of the structure predictions, turned out not to be important for AdoMet binding, suggesting that motif III has not been identified correctly. R128A and N130A, having mutations in the putative DNA binding domain, are unable to bind to DNA. Residues located in motifs IV, V, VI, and VIII are involved in catalysis (Asp193, Tyr196, Asp211, Ser229, Trp231, and Tyr258), some of them presumably in binding the flipped target base, because mutations at these residues fail to significantly interfere with DNA and AdoMet binding but strongly reduce catalysis. Our results are in substantial agreement with the structure prediction for EcoRV alpha-adenine-N6-methyltransferase and x-ray structures of other MTases.

Amino Acid Sequence↗
PubMed1998

Intra- vs intersubunit communication in the homodimeric restriction enzyme EcoRV: Thr 37 and Lys 38 involved in indirect readout are only important for the catalytic activity of their own subunit.

EcoRV is a dimer of two identical subunits which together form one binding site for the double-stranded DNA substrate. Concerted cleavage of both strands of the duplex requires intersubunit communication to synchronize the two catalytic centers of EcoRV. Here we address the question of how contacts to the DNA backbone trigger conformational changes which lead to the activation of both catalytic centers. The structure of the specific EcoRV-DNA complex shows that a region including amino acids Thr 37 and Lys 38 is involved in interactions with the DNA backbone and is a candidate for intersubunit communication. Homodimeric EcoRV T37A and K38A variants have a 1000-fold reduced catalytic activity. To examine whether Thr 37 and Lys 38 of one subunit affect the catalytic center in the same subunit and/or in the other subunit, we have produced heterodimeric variants containing a Thr 37 --> Ala or Lys 38 --> Ala substitution in one subunit combined with a wild type (wt) subunit (wt/T37A and wt/K38A) or with a subunit which contains an amino acid substitution (Asp 90 --> Ala) in the active site (D90A/T37A and D90A/K38A). Cleavage experiments with supercoiled pAT153 show that wt/T37A and wt/K38A preferentially nick the DNA. A steady-state kinetic analysis of the cleavage of an oligodeoxynucleotide substrate shows that the activity of wt/T37A and wt/K38A is half of that of wild type EcoRV, whereas D90A/T37A and D90A/K38A are almost inactive. These results demonstrate that Thr 37 and Lys 38 affect primarily the catalytic center in their own subunit and that both subunits of EcoRV can be activated independently of each other. We suggest that Thr 37 and Lys 38 control the catalytic activity of the active site in their own subunit by positioning alpha-helix B.

Catalysis↗
PubMed1998

Kinetic characterization of linear diffusion of the restriction endonuclease EcoRV on DNA.

We have examined the kinetic parameters of linear diffusion of EcoRV on DNA. The data were analyzed by Monte Carlo simulations in which the efficiency of recognition of EcoRV sites during linear diffusion, the efficiency of linear diffusion, and the behavior of enzymes at the ends of linear DNA is explicitly treated. The analysis of the dependence of linear diffusion on the concentrations of NaCl and MgCl2 shows that linear diffusion is maximal at 50 mM NaCl under all concentrations of MgCl2 tested and increases with increasing concentrations of Mg2+ up to 10 mM, the highest concentration used in the test. Under these conditions, EcoRV scans 2 x 10(6) bp during one binding event with a velocity of about 1.7 x 10(6) bp s-1. The enzyme tends to overlook cleavage sites at 1 mM but not at 10 mM MgCl2. This result confirms the thermodynamic finding that EcoRV does not bind very specifically to DNA in the absence of Mg2+. It demonstrates that there is a Mg2+-dependent continuous transition between a nonspecific and a specific binding mode of EcoRV to DNA. By comparing cleavage rates of linear DNA whose ends are free or blocked, we have shown that EcoRV has a very low probability to fall off at the ends of linear DNA. The enzyme rather is "reflected" and continues linear diffusion. EcoRV does not cleave oligonucleotides containing two EcoRV sites processively. Consequently, dissociation of the enzyme from the cleavage products is not preceded by a transfer to nonspecific DNA, and linear diffusion is not involved in product dissociation in EcoRV.

Base Sequence↗
PubMed1998

Kinetics of methylation and binding of DNA by the EcoRV adenine-N6 methyltransferase.

The EcoRV DNA methyltransferase (M.EcoRV) specifically methylates the first adenine within its recognition sequence GATATC. Methylation rates of DNA by this enzyme are strongly influenced by the length of oligonucleotide substrates employed. If substrates >20 bp compared to a 12mer substrate, the kcat/Km increases 100-fold, although the enzyme does not contact more than 12 base-pairs on the DNA. Single-turnover rates are higher than kcat values. M.EcoRV binding to DNA is fast but dissociation from the DNA is slow, demonstrating that the multiple-turnover rate is limited by the rate of product release. The kinetics of DNA binding by M.EcoRV are not in accordance with the thermodynamics binding constant, suggesting that the M.EcoRV-DNA complex is involved in a slow conformational change. The salt dependence of DNA binding is different for non-specific substrates (d ln(KAss)/d ln(cNaCl) = - 2, indicative of electrostatic interactions) and specific substrates (d ln(KAss)/d ln(cNaCl) = + 1, indicative of hydrophobic interactions). This result demonstrates that the M.EcoRV-DNA complex has a different conformation in both binding modes. M.EcoRV does not discriminate between hemimethylated and unmethylated substrates. Using the 20mer we have analyzed the temperature and pH dependence of the single-turnover rate constant of M.EcoRV-DNA methylation by M.EcoRV has an activation energy of 40 kJ/mol and its rate increases with increasing pH. The pH dependence reveals the presence of an ionizable residue with a pKa of 7.9, which must be unprotonated for catalysis. The rates of DNA methylation remain unchanged if an abasic site is introduced instead of the thymidine residue that is base-paired to the target adenine, demonstrating that flipping out the target adenine cannot contribute to the rate-limiting step of the enzymatic reaction.

Binding Sites↗
PubMed1998

The Flavobacterium okeanokoites adenine-N6-specific DNA-methyltransferase M.FokI is a tandem enzyme of two independent domains with very different kinetic properties.

The Flavobacterium okeanokoites adenine-N6-specific DNA-methyltransferase, M.FokI, modifies both adenine residues within its asymmetric recognition sequence 5'-GGATG/CATCC-3'. It is a fusion protein comprising two independent enzymes. We have cloned, overexpressed and purified full-length M.FokI as well as both individual domains and analyzed their kinetics of DNA methylation using unmethylated and hemimethylated oligodeoxynucleotide substrates. Our data show that both domains of M.FokI methylate DNA independently of each other but cooperate in DNA binding. In agreement to former studies, the N-terminal domain of M.FokI modifies the upper strand of the recognition sequence (GGATG). It strongly prefers hemimethylated (5'-GGATG/CmATCC-3'; mA = N6-methyladenosine) over unmethylated substrates. In contrast, the C-terminal domain prefers unmethylated DNA substrates. Surprisingly, in addition to methylating the lower strand of the recognition sequence (CATCC), M.FokI-(335-647) also modifies the upper strand (GGATG), albeit with a lower activity. In addition, methylation was detected at CACCC sites, but not at sites in which a central AT dinucleotide is flanked by at least one A x T or T x A base pair. These results suggests that M.FokI-(335-647) either has a very degenerate specificity for (G/C)AT(G/C) and sites similar to CATCC or GGATG or, alternatively, that it has a dual specificity for CATCC and GGATG.

Base Sequence↗
PubMed1998

Towards the design of rare cutting restriction endonucleases: using directed evolution to generate variants of EcoRV differing in their substrate specificity by two orders of magnitude.

The restriction endonuclease EcoRV cleaves DNA highly specifically within GATATC sequences. In order to create EcoRV variants that have an extended recognition site we have employed a semi-rational random mutagenesis/selection procedure. Twenty-two amino acid residues were subjected to random mutagenesis and about 500 EcoRV variants representing three generations of mutants were screened. Among these some highly active variants that strongly prefer AT-flanked cleavage sites (e.g. S183A/Q224R, T93S/I103F/S183A/T222S or N97T/S183A/T222S) and others that prefer GC flanks (e.g. K104N/A181T) were identified. As wild-type EcoRV does not discriminate between these cleavage sites, the generation of these variants represents a significant first step towards redesigning EcoRV to become an 8 or 10 bp cutter. Such enzymes, only very rarely found in nature, could be extremely helpful for the manipulation of large DNA fragments.

Deoxyribonucleases, Type II Site-Specific↗
PubMed1997

Does the restriction endonuclease EcoRV employ a two-metal-Ion mechanism for DNA cleavage?

Two models for the catalytic mechanism of the restriction endonuclease EcoRV exist which differ in the number and function of metal ions proposed to be directly involved in catalysis. In one model, two metal ions bound by Glu45, Asp74, and Asp90 are assumed to have a direct catalytic function; in the other, only one metal ion bound by Asp74 and Asp90. We show here that in the presence of Mn2+, the catalytic activity of an EcoRV-E45A mutant is only slightly reduced (1.8-fold) as compared to wild type EcoRV and that the single-turnover rate constant of DNA cleavage by E45A is reduced only 39-fold, whereas the D74A and D90A mutants are catalytically inactive under all conditions. These findings make an important catalytic function of Glu45, like binding of an essential divalent metal ion, unlikely. In addition, we have analyzed the dependence of the DNA cleavage rate by EcoRV and EcoRV mutants on the concentration of Mg2+ and Mn2+. We found for the wild type enzyme a sigmoidal dependence of the rate of DNA cleavage on the concentration of Mg2+ or Mn2+, indicative of at least two metal ions involved in DNA binding and catalysis. This, however, does not mean that EcoRV follows a two-metal-ion mechanism in DNA cleavage, because also for the E45A mutant a sigmoidal dependence of the rate of DNA cleavage on the Mg2+ concentration was found, making metal ion binding to the E45/D74 site unlikely. In contrast, the Y219C mutant shows a hyperbolic dependence. In agreement with results obtained earlier, these findings demonstrate binding of a Mg2+ ion at a site influenced by Tyr219, an amino acid residue that is far away from the active site. Metal binding at this site does not have a catalytic role but rather supports specific DNA binding. We conclude that on the basis of our data a two-metal-ion mechanism of DNA cleavage is unlikely for EcoRV and that the complex metal ion effects observed are due to metal ion binding at sites that are not directly involved in catalysis.

Bacteriophage lambda↗
PubMed1997

Recognition and cleavage of DNA by type-II restriction endonucleases.

Restriction endonucleases are enzymes which recognize short DNA sequences and cleave the DNA in both strands. Depending on the enzymological properties different types are distinguished. Type II restriction endonucleases are homodimers which recognize short palindromic sequences 4-8 bp in length and, in the presence of Mg2+, cleave the DNA within or next to the recognition site. They are capable of non-specific binding to DNA and make use of linear diffusion to locate their target site. Binding and recognition of the specific site involves contacts to the bases of the recognition sequence and the phosphodiester backbone over approximately 10-12 bp. In general, recognition is highly redundant which explains the extreme specificity of these enzymes. Specific binding is accompanied by conformational changes over both the protein and the DNA. This mutual induced fit leads to the activation of the catalytic centers. The precise mechanism of cleavage has not yet been established for any restriction endonuclease. Currently two models are discussed: the substrate-assisted catalysis mechanism and the two-metal-ion mechanism. Structural similarities identified between EcoRI, EcoRV, BamHI, PvuII and Cfr10I suggest that many type II restriction endonucleases are not only functionally but also evolutionarily related.

Binding Sites↗
PubMed1996

Linear diffusion of the restriction endonuclease EcoRV on DNA is essential for the in vivo function of the enzyme.

Linear diffusion along DNA is a mechanism of enhancing the association rates of proteins to their specific recognition sites on DNA. It has been demonstrated for several proteins in vitro, but to date in no case in vivo. Here we show that the restriction endonuclease EcoRV slides along the DNA, scanning approximately 1000 bp in one binding event. This process is critically dependent on contacts between amino acid residues of the protein and the backbone of the DNA. The disruption of single hydrogen bonds and, in particular, the alteration of electrostatic interactions between amino acid side chains of the protein and phosphate groups of the DNA interfere with or abolish effective sliding. The efficiency of linear diffusion is dependent on salt concentration, having a maximum at 50 mM NaCl. These results suggest that a nonspecific and mobile binding mode capable of linear diffusion is dependent on a subtle balance of forces governing the interaction of the enzyme and the DNA. A strong correlation between the ability of EcoRV mutants to slide along the DNA in vitro and to protect Escherichia coli cells from phage infection demonstrates that linear diffusion occurs in vivo and is essential for effective phage restriction.

DNA↗
PubMed1996

Introduction of asymmetry in the naturally symmetric restriction endonuclease EcoRV to investigate intersubunit communication in the homodimeric protein.

Type II restriction endonucleases are dimers of two identical subunits that together form one binding site for the double-stranded DNA substrate. Cleavage within the palindromic recognition site occurs in the two strands of the duplex in a concerted manner, due to the action of two catalytic centers, one per subunit. To investigate how the two identical subunits of the restriction endonuclease EcoRV cooperate in binding and cleaving their substrate, heterodimeric versions of EcoRV with different amino acid substitutions in the two subunits were constructed. For this purpose, the ecorV gene was fused to the coding region for the glutathione-binding domain of the glutathione S-transferase and a His6-tag, respectively. Upon cotransformation of Escherichia coli cells with both gene fusions stable homo- and heterodimers of the EcoRV variants are produced, which can be separated and purified to homogeneity by affinity chromatography over Ni-nitrilotriacetic acid and glutathione columns. A steady-state kinetic analysis shows that the activity of a heterodimeric variant with one inactive catalytic center is decreased by 2-fold, demonstrating that the two catalytic centers operate independently from each other. In contrast, heterodimeric variants with a defect in one DNA-binding site have a 30- to 50-fold lower activity, indicating that the two subunits of EcoRV cooperate in the recognition of the palindromic DNA sequence. By combining a subunit with an inactive catalytic center with a subunit with a defect in the DNA-binding site, EcoRV heterodimers were produced that only nick DNA specifically within the EcoRV recognition sequence.

Base Sequence↗
PubMed1996

Probing the indirect readout of the restriction enzyme EcoRV. Mutational analysis of contacts to the DNA backbone.

According to the crystal structure of the specific EcoRV.DNA complex, not only the functional groups of the nucleobases but also the phosphate groups of the DNA backbone are contacted by the enzyme. To examine the contribution of backbone contacts to substrate recognition and catalysis by EcoRV, we exchanged 12 amino acids residues located close to phosphate groups by site-directed mutagenesis. We purified the resulting EcoRV mutants and characterized them with respect to their DNA binding and cleavage activity. According to our steady state kinetic analysis, there are strong interactions between three basic amino acid residues (Lys-119, Arg-140, and Arg-226) and the phosphate backbone that support specific binding presumably by inducing and maintaining the kinked conformation of the DNA observed in the specific EcoRV.DNA complex. These contacts are important in both the ground state and the transition state. Other, uncharged residues (Thr-93 and Ser-112), which could be involved in hydrogen bonds to the phosphate groups, are needed primarily to stabilize the transition state. An especially important amino acid residue is Thr-37, which seems to couple recognition to catalysis by indirect readout.

Amino Acid Sequence↗
PubMed1996

Horizontal gene transfer contributes to the wide distribution and evolution of type II restriction-modification systems.

Restriction modification (RM) systems serve to protect bacteria against bacteriophages. They comprise a restriction endonuclease activity that specifically cleaves DNA and a corresponding methyltransferase activity that specifically methylates the DNA, thereby protecting it from cleavage. Such systems are very common in bacteria. To find out whether the widespread distribution of RM systems is due to horizontal gene transfer, we have compared the codon usages of 29 type II RM systems with the average codon usage of their respective bacterial hosts. Pronounced deviations in codon usage were found in six cases: EcoRI, EcoRV, KpnI, SinI, SmaI, and TthHB81. They are interpreted as evidence for horizontal gene transfer in these cases. As the methodology is expected to detect only one-fourth to one-third of all horizontal gene transfer events, this result implies that horizontal gene transfer had a considerable influence on the distribution and evolution of RM systems. In all of these six cases the codon usage deviations of the restriction enzyme genes are much more pronounced than those of the methyltransferase genes. This result suggests that in these cases horizontal gene transfer had occurred sequentially with the gene for the methyltransferase being first acquired by the cell. This can be explained by the fact that an active restriction endonuclease is highly toxic in cells whose DNA is not protected from cleavage by a corresponding methyltransferase.

Bacteria↗