Biomedical subjects

A Pingoud

Publications and source records attributed to A Pingoud.

At least 55 records · Page 3Linked to original sources

PubMed1998

Biochemical characterization of Anabaena sp. strain PCC 7120 non-specific nuclease NucA and its inhibitor NuiA.

We have established overexpression systems and purification protocols for NucA and NuiA, a sugar non-specific nuclease and its protein inhibitor from Anabaena sp. strain PCC 7120, in order to characterize these proteins in detail. CD spectroscopy revealed that NucA has a similar secondary-structure composition, 13% alpha helix and 20% beta sheet, to the related Serratia nuclease, while NuiA represents a protein with a higher alpha-helical (29%) and beta-sheet (24%) content than NucA. Denaturation experiments showed that the stabilities of NucA and NuiA are in the typical range for proteins of mesophilic organisms, NuiA with deltaG0H2O = 63.4 J x mol(-1)residue, being slightly more stable than its target NucA with delta deltaG0H2O = 46.3 J x mol(-1)residue. The nuclease requires divalent metal ions as cofactors, the optimum concentration being around 5 mM for Mn2+ or Mg2+. The order of effectiveness of various divalent cations to function as cofactors for the hydrolytic activity of NucA is Mn2+ = Co2+ > Mg2+ > or = Ni2+ >> or Ca2+ = Cd2+ at a concentration of 5 mM. Nuclease activity decreases with increasing concentration of monovalent salt. The activity of NucA shows a pH optimum at pH 5.5-7.5. The temperature optimum is around 35 degrees C, the activation energy was calculated to be 53 kJ mol(-1). The specific activity of the nuclease towards high molecular-mass DNA is 8.4 x 10(6) Kunitz-units x mg(-1), which means that NucA is one of the most active nucleases known. Kinetic constants for the cleavage of various DNA and RNA substrates by NucA are all in the range Km < or = 0.1 mg x ml(-1) and k(cat) approximately 1000 s(-1). As other non-specific nucleases, NucA exhibits sequence preferences, similar to the related Serratia nuclease, NucA avoids cleavage of d(A) x d(T) tracts. The nucleolytic activity of NucA is completely inhibited at equimolar concentrations of nuclease and inhibitor. An ultracentrifugation analysis showed that NucA and NuiA form a 1:1 complex. The interaction of NucA with NuiA was also investigated by CD spectroscopy and revealed no major conformational changes upon complex formation of the two proteins.

Amino Acid 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

Characterization of the interaction between the restriction endonuclease McrBC from E. coli and its cofactor GTP.

McrBC, a GTP-dependent restriction enzyme from E. coli K-12, cleaves DNA containing methylated cytosine residues 40 to 80 residues apart and 3'-adjacent to a purine residue (PumCN40-80PumC). The presence of the three consensus sequences characteristic for guanine nucleotide binding proteins in one of the two subunits of McrBC suggests that this subunit is responsible for GTP binding and hydrolysis. We show here that (i) McrB binds GTP with an affinity of 10(6) M-1 and that GTP binding stabilizes McrB against thermal denaturation. (ii) McrB binds GDP about 50-fold and ATP at least three orders of magnitude more weakly than GTP. (iii) McrB hydrolyzes GTP in the presence of Mg2+ with a steady-state rate of approximately 0.5 min-1. (iv) McrC stimulates GTP hydrolysis 30-fold, but substrate DNA has no detectable effect on the GTPase activity of McrB, neither by itself nor in the presence of McrC. (v) Substitution of N339 and N376 with alanine allowed us to identify NTAD (339 to 342) rather than NKKA (376 to 379) as the equivalent of the third consensus sequence motif characteristic for guanine nucleotide binding proteins, NKXD.

Adenosine Triphosphate↗

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↗

PubMed1997

The recognition of methylated DNA by the GTP-dependent restriction endonuclease McrBC resides in the N-terminal domain of McrB.

McrBC is a GTP-dependent restriction endonuclease of E. coli K12, selectively directed against DNA containing modified cytosine residues. McrB, one of its components, is responsible for the binding and, together with McrC, for the cleavage of DNAs containing two 5'-Pu(m)C sites separated by 40-80 base pairs. Gel retardation assays with wild-type and mutant McrB reveal that (i) single 5'-Pu(m)C sites in DNA can be sufficient to elicite binding by McrB. Binding to such substrates is, however, weak and strongly dependent on the sequence context of Pu(m)C sites. (ii) Strong DNA binding (K(ass) approximately 10(7)M[-1]) is dependent on the presence of at least two Pu(m)C sites, even if they are separated by less than 40 bp, and is modulated by the sequence context (-A(m)CCGGT- --> -A(m)CT(C/G)AGT- --> -AGG(m)CCT- --> -AAG(m)CTT-). (iii) DNA binding by McrB is accompanied by formation of distinct multiple complexes whose distribution is modulated by GTP. (iv) McrC, which cannot bind DNA by itself, moderately stimulates the DNA binding of McrB and converts McrB-DNA complexes to large aggregates. (v) Deletion of the C-terminal half of McrB, which harbors the three consensus sequences characteristic for guanine nucleotide binding proteins, leads to protein inactive in GTP binding and/or hydrolysis and in McrC-assisted DNA cleavage; the protein, however, remains fully competent in DNA binding. (vi) Mutations in McrB which lead to a reduction in GTP binding and/or hydrolysis can affect DNA binding, suggesting that the two activities are coupled in the full-length protein.

Base Composition↗

PubMed1996

Analysis of the reaction mechanism of the non-specific endonuclease of Serratia marcescens using an artificial minimal substrate.

We have studied the mechanism of action of the Serratia nuclease using deoxythymidine 3',5'-bis-(p-nitrophenyl-phosphate) as a substrate. A comparison of the activity with which the wild-type enzyme and several mutant enzymes attack this artificial substrate and herring sperm DNA, respectively, supports the suggestion that His89 is the general base and a Mg2+ ion bound to Glu127 the general acid in the mechanism of phosphodiester bond hydrolysis by the Serratia nuclease, and that Asn119 directly participates in catalysis, for example by transition state stabilisation. Arg57, Arg87 and Arg131, essential for nuclease activity, are not needed for cleavage of the artificial substrate, suggesting that they are involved in binding and positioning of nucleic acid substrates.

DNA↗

PubMed1996

Binding, bending and cleavage of DNA substrates by the homing endonuclease Pl-SceI.

To characterize the interaction between the homing endonuclease PI-SceI and DNA, we prepared different DNA substrates containing the natural recognition sequence or parts thereof. Depending on the nature of the substrates, efficient cleavage is observed with a DNA containing approximatel 30 bp of the natural recognition sequence using supercoiled plasmids, approximately 40-50 bp using linearized plasmids and > 50 bp using synthetic double-stranded oligodeoxynucleotides. Cleavage of supercoiled plasmids occurs without accumulation of the nicked intermediate. In the presence of Mn2+, DNA cleavage by PI-SceI is more efficient than with Mg2+ and already occurs with substrates containing a shorter part of the recognition sequence. The requirements for strong binding are less stringent: a 35 bp oligodeoxynucleotide which is not cleaved is bound as firmly as other longer oligodeoxynucleotides. PI-SceI binds with high affinity to one of its cleavage products, a finding which may explain why PI-SceI hardly shows enzymatic turnover in vitro. Upon binding, two complexes are formed, which differ in the degree of bending (45 degrees versus 75 degrees). According to a phasing analysis bending is directed into the major groove. Strong binding, not, however, cleavage is also observed with the genetically engineered enzymatically inactive variant comprising amino acids 1-277. Models for binding and cleavage of DNA by PI-SceI are discussed based on these results.

Binding Sites↗

PubMed1996

Kinetic analysis of the cleavage of natural and synthetic substrates by the Serratia nuclease.

The extracellular nuclease from Serratia marcescens is a non-specific endonuclease that hydrolyzes double-stranded and single-stranded DNA and RNA with high specific activity. Steady-state and presteady-state kinetic cleavage experiments were performed with natural and synthetic DNA and RNA substrates to understand the mechanism of action of the Serratia nuclease. Most of the natural substrates are cleaved with similar Kcat and K(m) values, the Kcat/K(m) ratios being comparable to that of staphylococcal nuclease. Substrates with extreme structural features, like poly(dA).poly(dT) or poly(dG).poly(dC), are cleaved by the Serratia nuclease with a 50 times higher or 10 times lower K(m), respectively, as salmon testis DNA. Neither with natural DNA or RNA nor synthetic oligodeoxynucleotide substrates did we observe substrate inhibition for the Serratia nuclease as reported recently. Experiments with short oligodeoxynucleotides confirmed previous results that for moderately good cleavage activity the substrate should contain at least five phosphate residues. Shorter substrates are still cleaved by the Serratia nuclease, albeit at a rate reduced by a factor of more than 100. Cleavage experiments with oligodeoxynucleotides substituted by a single phosphorothioate group showed that the negative charge of the pro-Rp-oxygen of the phosphate group 3' adjacent to the scissile phosphodiester bond is essential for cleavage, as only the Rp-phosphorothioate supports cleavage at the 5' adjacent phosphodiester bond. Furthermore, the modified bond itself is only cleaved in the Rp-diastereomer, albeit 1000 times more slowly than the corresponding unmodified phosphodiester bond, which offers the possibility to determine the stereochemical outcome of cleavage. Pre-steady-state cleavage experiments demonstrate that it is not dissociation of products but association of enzyme and substrate or the cleavage of the phosphodiester bond that is the rate-limiting step of the reaction. Finally, it is shown that Serratia nuclease accepts thymidine 3',5'-bis(p-nitrophenyl)phosphate as a substrate and cleaves it at its 5'-end to produce nitrophenol and thymidine 3'-(p-nitrophenylphosphate) 5-phosphate. The rate of cleavage of this artificial substrate, however, is 6-7 orders of magnitude smaller than the rate of cleavage of macromolecular DNA or RNA.

Base Sequence↗

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

A quantitative microtiter plate nuclease assay based on ethidium/DNA fluorescence.

A microtiter plate assay was developed to quantitate the nuclease activity of the extracellular Serratia marcescens endonuclease under different buffer conditions. Substrate cleavage was followed as decrease in ethidium/DNA fluorescence using a uv-transilluminator and a video documentation system. Time courses of DNA cleavage were recorded and cleavage rates determined very precisely within a factor of 1.2. The assay has a linear dynamic range covering three orders of magnitude of nuclease activity and can be carried out very quickly within a few minutes. It can also be used with RNA as substrate. With appropriate modifications it should be possible to adapt this assay for other enzymatic reactions which are accompanied by changes in absorbance or fluorescence.

Animals↗

PubMed1996

Analysis of the mechanism of the Serratia nuclease using site-directed mutagenesis.

Based on crystal structure analysis of the Serratia nuclease and a sequence alignment of six related nucleases, conserved amino acid residues that are located in proximity to the previously identified catalytic site residue His89 were selected for a mutagenesis study. Five out of 12 amino acid residues analyzed turned out to be of particular importance for the catalytic activity of the enzyme: Arg57, Arg87, His89, Asn119 and Glu127. Their replacement by alanine, for example, resulted in mutant proteins of very low activity, < 1% of the activity of the wild-type enzyme. Steady-state kinetic analysis of the mutant proteins demonstrates that some of these mutants are predominantly affected in their kcat, others in their Km. These results and the determination of the pH and metal ion dependence of selected mutant proteins were used for a tentative assignment for the function of these amino acid residues in the mechanism of phosphodiester bond cleavage by the Serratia nuclease.

Amino Acid Sequence↗

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↗

PubMed1996

Engineering novel restriction endonucleases: principles and applications.

Restriction endonucleases cleave DNA with remarkable sequence specificity. In this review, we summarize the status of, and prospects for, engineering restriction endonucleases with new specificities. Such variants could be of considerable commercial value because restriction enzymes are among the most frequently used enzymes in molecular biology, and not all the desirable specificities are available. While it has not yet been possible to effect specificity changes, mutant have been described that (1) exhibit relaxed specificity, (2) favour modified substrates over their natural substrates, (3) discriminate between cleavage sites located in different sequences, (4) prefer metal ions other than Mg2+ as cofactors for cleavage, or (5) possess site-specific DNA-nicking activity.

Amino Acid Sequence↗

PubMed1996

EcoRV-T94V: a mutant restriction endonuclease with an altered substrate specificity towards modified oligodeoxynucleotides.

Synthetic oligodeoxynucleotides with single methyl phosphonate (mp) substitutions were used for an analysis of the contribution of phosphate contacts to the recognition of the cleavage site by the restriction endonuclease EcoRV. Only in the last position within the recognition sequence, is the methyl phosphonate substitution tolerated by the enzyme. The wild-type enzyme cleaves the Sp diastereomer of the oligodeoxynucleotide GACGATATmpCGTC and the unmodified sequence with equal rates, whereas the Rp diastereomer is cleaved much more slowly. Inspection of the crystal structure of an EcoRV-DNA complex revealed that the non-bridging oxygen atoms of the phosphodiester bond between the T and C bases are in hydrogen bonding distance of the hydroxyl group of the amino acid Thr94. We therefore tried to engineer a variant of EcoRV that would prefer a methyl phosphonate linkage over a normal phosphodiester bond and produced mutants with amino acid exchanges at position 94. One of them, Thr94Val, shows a dramatically reduced activity towards the unmodified DNA and does not accept the Rp diastereomer, but cleaves the Sp diastereomer with the same rate as wild-type EcoRV. Its selectivity, i.e. the ratio of cleavage rates determined for the unmodified and modified substrates, differs by three orders of magnitude from that of the wild-type enzyme.

DNA↗

PubMed1996

Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants.

The EcoRV DNA methyltransferase (M.EcoRV) is an alpha-adenine methyltransferase. We have used two different programs to predict the secondary structure of M.EcoRV. The resulting consensus prediction was tested by a mutant profiling analysis. 29 neutral mutations of M.EcoRV were generated by five cycles of random mutagenesis and selection for active variants to increase the reliability of the prediction and to get a secondary structure prediction for some ambiguously predicted regions. The predicted consensus secondary structure elements could be aligned to the common topology of the structures of the catalytic domains of M.HhaI and M.TaqI. In a complementary approach we have isolated nine catalytically inactive single mutants. Five of these mutants contain an amino acid exchange within the catalytic domain of M.EcoRV (Val2-Ala, Lys81Arg, Cys192Arg, Asp193Gly, Trp231Arg). The Trp231Arg mutant binds DNA similarly to wild-type M.EcoRV, but is catalytically inactive. Hence this mutant behaves like a bona fide active site mutant. According to the structure prediction, Trp231 is located in a loop at the putative active site of M.EcoRV. The other inactive mutants were insoluble. They contain amino acid exchanges within the conserved amino acid motifs X, III or IV in M.EcoRV confirming the importance of these regions.

Amino Acid Sequence↗