Mung bean (Phaseolus aureus) nuclease. A mechanistic investigation of the DNA-cleavage reaction using a dinucleoside phosphorothioate.

Mung-bean (Phaseolus aureus) nuclease has been found to cleave the Sp diastereoisomer of 5'-O-thymidyl 3'-O-(2'-deoxyadenosyl)phosphorothioate, (Sp)-d[Ap(S)T], in 18O-labelled water with inversion of configuration at phosphorus to give (Sp)-thymidine 5'-[16O, 18O]phosphorothioate, the stereochemistry of which was deduced by methylation to (Rp,Sp)-thymidine 5'-S-methyl-O-methyl-[16O,18O]phosphorothioate and 31P-n.m.r. analysis. This result is consistent with a mechanism involving a direct 'in-line' attack of water on DNA for the nuclease-catalysed reaction without the involvement of a covalent nucleotidylated-enzyme intermediate.     

The stereochemical course of the restriction endonuclease EcoRI-catalyzed reaction

The restriction endonuclease EcoRI hydrolyzes the Rp diastereomer of d(pGGsAATTCC), an analogue of d(pGGAATTCC) containing a chiral phosphorothioate group at the cleavage site between the deoxyguanosine and the deoxyadenosine residues (Connolly, B.A., Potter, B.V.L., Eckstein, F., Pingoud, A., and Grotjahn, L. (1984) Biochemistry 23, 3343-3453). Performing the reaction in H2(18)O leads to d(pGG) and the hexanucleotide d([18O, S]pAATTCC) which has an 18O-containing phosphorothioate group at the 5' terminus. Further hydrolysis of this hexamer with nuclease P1 yields deoxyadenosine 5'-O-[18O]phosphorothioate which can be stereospecifically phosphorylated with adenylate kinase and pyruvate kinase to give Sp-[18O] deoxyadenosine 5'-O-(1-thiotriphosphate). 31P NMR spectroscopy shows the oxygen-18 in this compound to be in a bridging position between the alpha- and beta-phosphorus atoms. Thus, the hydrolysis reaction catalyzed by EcoRI proceeds with inversion of configuration at phosphorus. This result is compatible with a direct enzyme-catalyzed nucleophilic attack of H2O at phosphorus without involvement of a covalent enzyme intermediate.     

Stereochemical course of DNA hydrolysis by nuclease S1

Nuclease S1 hydrolyzes the Sp-diastereomer of 5'-O-(2'-deoxyadenosyl)-3'-O-thymidyl phosphorothioate in H2(18)O to [18O]deoxyadenosine 5'-O-phosphorothioate which can be phosphorylated enzymatically to the Sp-diastereomer of [alpha-18O]deoxyadenosine 5'-O-(1-thiotriphosphate). 31P nmr spectroscopy shows the oxygen-18 in this compound to be in a nonbridging position at the alpha-phosphorus, indicating that the hydrolysis reaction catalyzed by nuclease S1 proceeds with inversion of configuration at phosphorus. This result is compatible with a direct nucleophilic attack of H2O at phosphorus without the involvement of a covalent enzyme intermediate.     

Electrostatic Suppression Allows Tyrosine Site-specific Recombination in the Absence of a Conserved Catalytic Arginine

The active site of the tyrosine family site-specific recombinase Flp contains a conserved catalytic pentad that includes two arginine residues, Arg-191 and Arg-308. Both arginines are essential for the transesterification steps of strand cleavage and strand joining in DNA substrates containing a phosphate group at the scissile position. During strand cleavage, the active site tyrosine supplies the nucleophile to form a covalent 3′-phosphotyrosyl intermediate. The 5′-hydroxyl group produced by cleavage provides the nucleophile to re-form a 3′-5′ phosphodiester bond in a recombinant DNA strand. In previous work we showed that substitution of the scissile phosphate (P) by the charge neutral methylphosphonate (MeP) makes Arg-308 dispensable during the catalytic activation of the MeP diester bond. However, in the Flp(R308A) reaction, water out-competes the tyrosine nucleophile (Tyr-343) to cause direct hydrolysis of the MeP diester bond. We now report that for MeP activation Arg-191 is also not required. In contrast to Flp(R308A), Flp(R191A) primarily mediates normal cleavage by Tyr-343 but also exhibits a weaker direct hydrolytic activity. The cleaved MeP-tyrosyl intermediate formed by Flp(R191A) can be targeted for nucleophilic attack by a 5′-hydroxyl or water and channeled toward strand joining or hydrolysis, respectively. In collaboration with wild type Flp, Flp(R191A) promotes strand exchange between MeP- and P-DNA partners. Loss of a catalytically crucial positively charged side chain can thus be suppressed by a compensatory modification in the DNA substrate that neutralizes the negative charge on the scissile phosphate.     

The stereochemical course of phospho transfer catalyzed by adenylosuccinate synthetase. A reaction pathway via a phosphorylated intermediate with net inversion

The stereochemical course of phospho transfer in the reaction catalyzed by adenylosuccinate synthetase from rat muscle has been determined with chiral [gamma-17O,18O]GTP gamma S as a substrate. The stereochemical configuration of the product, inorganic thiophosphate, was determined by 31P NMR after the compound was stereospecifically incorporated into ATP beta S. The reaction goes with net inversion of configuration, which is the course for a single phospho transfer, even though 6-phospho-IMP is probably an intermediate on the normal reaction pathway (Liebermann, I. (1956) J. Biol. Chem. 223, 327-339). The breakdown of this intermediate goes by C-O bond cleavage and so is not a true phospho transfer step. Thus, inversion of configuration during the course of this ligase reaction is consistent with a single phospho transfer step in the overall reaction, the formation of the phosphorylated intermediate.     

Inversion of the phosphate chirality at the target site of Mu DNA strand transfer: evidence for a one-step transesterification mechanism.

Central to transposition of phage Mu are two reactions mediated by the MuA protein. First, MuA introduces single-stranded cuts at the ends of the Mu DNA to generate 3' OH termini. In the subsequent strand-transfer step, the MuA-Mu DNA end complex cuts a target DNA and joins the Mu 3' ends to the 5' ends of the target. DNA containing chiral phosphorothioates was used to demonstrate inversion of the chirality during the course of strand transfer. This result strongly supports a one-step transesterification mechanism in which the 3' OH of the cleaved donor DNA is the attacking nucleophile. Furthermore, this donor 3' OH group was essential for target DNA cleavage. In contrast, during lambda integration the phosphate chirality was retained, as expected for a two-step transesterification involving a covalent protein-DNA intermediate.     

Stereochemical selectivity of group II intron splicing, reverse splicing, and hydrolysis reactions.

We have previously shown, using phosphorothioate substitutions at splice site, that both transesterification steps of group II intron self-splicing proceed, by stereochemical inversion, with an Sp but not an Rp phosphorothioate. Under alternative reaction conditions or with various intron fragments, group II introns can splice following hydrolysis at the 5' splice site and can also hydrolyze the bond between spliced exons (the spliced-exon reopening reaction). In this study, we have determined the stereochemical specificities of all of the major model hydrolytic reactions carried out by the aI5 gamma intron from Saccharomyces cerevisiae mitochondria. For all substrates containing exon 1 and most of the intron, the stereospecificity of hydrolysis is the same as for the step 1 transesterification reaction. In contrast, the spliced-exon reopening reaction proceeds with an Rp but not an Sp phosphorothioate at the scissile bond, as does true reverse splicing. Thus, by stereochemistry, this reaction appears to be related to the reverse of step 2 of self-splicing. Finally, a substrate RNA that contains the first exon and nine nucleotides of the intron, when reacted with the intron ribozyme, releases the first exon regardless of the configuration of the phosphorothioate at the 5' splice site, suggesting that this substrate can be cleaved by either the step 1 or the step 2 reaction site. Our findings clarify the relationships of these model reactions to the transesterification reactions of the intact self-splicing system and permit new studies to be interpreted more rigorously.     

Stereochemistry of cleavage of internucleotide bonds by Serratia marcescens endonuclease.

Endonuclease from Serratia marcescens hydrolyzes internucleotide phosphorothioate linkages of R(P) configuration with inversion of configuration at P-atom. This observation supports a reported architecture of the active site, with 3'-bridging and pro-S(P) non-bridging oxygen atoms of the scissile phosphate group involved in direct contact with hydrated magnesium cation, while His-89 activates a water molecule which attacks the phosphorus atom according to a one-step in-line mechanism. The presence of a phosphorothioate bond of S(P) configuration downstream to that one being cleaved reduces the rate of hydrolysis. This suggests participation of the pro-S(P) oxygen atom of that phosphate bond in the mechanism of action of the enzyme, which was not detected in published crystallographic analyses.     

Changing endonuclease EcoRII Tyr308 to Phe abolishes cleavage but not recognition: possible homology with the Int-family of recombinases.

Endonuclease EcoRII is one of a group of type II restriction enzymes, including Nael, Narl, BspMI, HpaII, and SacII, that require binding of an enhancer sequence to cleave DNA. Comparison of the EcoRII amino-acid sequence with the amino-acid consensus motifs that differentiate between recombinase families uncovered similarity between a 29 amino-acid sequence in the carboxyl end of EcoRII and the motif defining the integrase family of recombinases. This similarity implied that EcoRII tyrosine 308 should be involved in catalyzing hydrolysis of the scissile bond. Site-directed mutagenesis was used to mutate Tyr308 to Phe. The phenylalanine-substituted enzyme could not cleave T5 DNA under conditions in which wild-type enzyme completely cleaved this DNA. The Tyr308 to Phe mutation abolished cleavage activity but not specific binding to DNA. No evidence was found for the existence during the cleavage reaction of a covalent linkage between Tyr308 and DNA.     

Binding and cleavage of nucleic acids by the "hairpin" ribozyme

The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+.     

Modification of Escherichia coli DNA ligase by cleavage with trypsin.

Limited treatment of Escherichia coli DNA ligase with trypsin results in rapid loss of DNA joining activity. However, the ability to react with DPN to form the covalent enzyme-AMP intermediate is unaffected. The cleaved enzyme is also unable to catalyze the formation of DNA-adenylate, the second covalent intermediate in the ligase-catalyzed reaction. These findings demonstrate that portions of the DNA ligase molecule that are required for phosphodiester bond formation are not required for at least one of the partial reactions catalyzed by this enzyme.     

Stereochemical course of the hydrolysis of thymidine 5'-(4-nitrophenyl [17O,18O]phosphate) in H216O catalyzed by the phosphodiesterase from snake venom

The phosphodiesterase from snake venom catalyzes the hydrolysis of the Rp diastereomer of thymidine 5'-(4-nitrophenyl [17O,18O]phosphate) in H216O with retention of configuration at phosphorus. This result is in agreement with those previously reported for the hydrolysis of chiral phosphorothioate substrates (Bryant, F. R., and Benkovic, S. J. (1979) Biochemistry 18, 2825-2828; Burgers, P. M. J., Eckstein, F., and Hunneman, D. H. (1979) J. Biol. Chem. 254, 7476-7478). The hydrolysis reaction catalyzed by this enzyme occurs via formation of a covalent nucleotidylated enzyme intermediate.     

The stereochemical course of the ribulose-5-phosphate kinase-catalyzed reaction

Spinach-leaf ribulose-5-phosphate kinase catalyzes the reaction of (Rp)-[beta, gamma-18O, gamma-18O]adenosine 5'-(3-thiotriphosphate) with ribulose 5-phosphate to form ribulose 1-[18O]phosphorothioate 5-phosphate. This product is incubated with CO2, Mg2+, and ribulose-bisphosphate carboxylase to form the [18O]phosphorothioate of D-glycerate. Reduction of this material using phosphoglycerate kinase/ATP, glyceraldehyde-3-phosphate dehydrogenase/NADH, triose-phosphate isomerase, and glycerol-phosphate dehydrogenase/NADH produces glycerol 3-[18O]phosphorothioate, which is subjected to ring closure using diethylphosphorochloridate. This in-line reaction produces a diastereoisomeric mixture of glycerol 2,3-cyclic phosphorothioates. 31P NMR spectroscopy was used to analyze the 18O content of the products. The anti-diastereoisomer, which is the major isomer formed and corresponds to the downfield 31P NMR signal (Pliura, D.H., Schomburg, D., Richard, J.P., Frey, P.A., and Knowles, J.R. (1980) Biochemistry 19, 325-329), retains the 18O label. This observation indicates that the ribulose-5-phosphate kinase reaction proceeds with inversion of configuration at phosphorus. The reaction is, therefore, unlikely to involve the participation of a covalent phosphoryl-enzyme intermediate.     

Stereochemical outcome of the hydrolysis reaction catalyzed by the EcoRV restriction endonuclease.

The stereochemical course of the reaction catalyzed by the EcoRV restriction endonuclease has been determined. This endonuclease recognizes GATATC sequence and cuts between the central T and dA bases. The Rp isomer of d(GACGATsATCGTC) (this dodecamer contains a phosphorothioate rather than the usual phosphate group between the central T and dA residues, indicated by the s) was a substrate for the endonuclease. Performing this reaction in H2 18O gave [18O]dps(ATCGTC) (a pentamer containing an 18O-labeled 5'-phosphorothioate) which was converted to [18O]dAMPS with nuclease P1. This deoxynucleoside 5'-[18O]phosphorothioate was stereospecifically converted to [18O]dATP alpha S with adenylate kinase and pyruvate kinase [Brody, R. S., & Frey, P. A. (1981) Biochemistry 20, 1245-1251]. Analysis of the position of the 18O in this product by 31P NMR spectroscopy showed that it was in a bridging position between the alpha- and beta-phosphorus atoms. This indicates that the EcoRV hydrolysis proceeds with inversion of configuration at phosphorus. The simplest interpretation is that the mechanism of this endonuclease involves a direct in-line attack at phosphorus by H2O with a trigonal bipyramidal transition state. A covalent enzyme oligodeoxynucleotide species can be discounted as an intermediate. An identical result has been previously observed with the EcoR1 endonuclease [Connolly, B. A., Eckstein, F., & Pingoud, A. (1984) J. Biol. Chem. 259, 10760-10763]. X-ray crystallography has shown that both of these endonucleases contain a conserved array of amino acids at their active sites. Possible mechanistic roles for these conserved amino acids in the light of the stereochemical findings are discussed.     

Coupling of fast and slow modes in the reaction pathway of the minimal hammerhead ribozyme cleavage

By employing classical molecular dynamics, correlation analysis of coupling between slow and fast dynamical modes, and free energy (umbrella) sampling using classical as well as mixed quantum mechanics molecular mechanics force fields, we uncover a possible pathway for phosphoryl transfer in the self-cleaving reaction of the minimal hammerhead ribozyme. The significance of this pathway is that it initiates from the minimal hammerhead crystal structure and describes the reaction landscape as a conformational rearrangement followed by a covalent transformation. The delineated mechanism is catalyzed by two metal (Mg(2+)) ions, proceeds via an in-line-attack by CYT 17 O2' on the scissile phosphorous (ADE 1.1 P), and is therefore consistent with the experimentally observed inversion configuration. According to the delineated mechanism, the coupling between slow modes involving the hammerhead backbone with fast modes in the cleavage site appears to be crucial for setting up the in-line nucleophilic attack.     

Synthesis and configurational analysis of a dinucleoside phosphate isotopically chiral at phosphorus. Stereochemical course of Penicillium citrum nuclease P1 reaction

(Rp)- and (Sp)-5'-O-thymidyl 3'-O-thymidyl [18O]phosphates have been synthesized by reaction of the respective (Sp)- and (Rp)-phosphorothioate precursors with N-bromosuccinimide in dioxane and H218O. Stereochemical analysis of the product derived from the (Rp)-phosphorothioate by digestion with snake venom phosphodiesterase in H217O and examination of the isotopic chirality of the resulting thymidine 5'-[16O,17O,18O]phosphate demonstrate that the replacement reaction has proceeded with inversion of configuration at phosphorus. Inspection of the 31P NMR spectrum of the methyl esters prepared from (Sp)-5'-O-thymidyl 3'-O-thymidyl [18O]phosphate confirms that the replacement reaction has proceeded with very little if any racemization. This spectrum also allows the assignment of the absolute configuration of these methyl triesters. (Rp)-5'-O-Thymidyl 3'-O-thymidyl [18O]phosphate has been used to demonstrate that the stereochemical course of the hydrolytic reaction catalyzed by nuclease P1 from Penicillium citrum proceeds with inversion of configuration at phosphorus and therefore probably does not involve the participation of a covalent enzyme intermediate.     

DNA strand transfer reactions catalyzed by vaccinia topoisomerase: hydrolysis and glycerololysis of the covalent protein-DNA intermediate.

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at sites containing the sequence 5'-CCCTT. The T nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that the enzyme catalyzes hydrolysis of the covalent intermediate, resulting in formation of a 3'-phosphate-terminated DNA cleavage product. The hydrolysis reaction is pH-dependent (optimum pH = 9.5) and is slower, by a factor of 10(-5), than the rate of topoisomerase-catalyzed strand transfer to a 5'-OH terminated DNA acceptor strand. Mutants of vaccinia topoisomerase containing serine or threonine in lieu of the active site Tyr-274 form no detectable covalent intermediate and catalyze no detectable DNA hydrolysis. This suggests that hydrolysis occurs subsequent to formation of the covalent protein-DNA adduct and not via direct attack by water on DNA. Vaccinia topoisomerase also catalyzes glycerololysis of the covalent intermediate. The rate of glycerololysis is proportional to glycerol concentration and is optimal at pH 9.5.     

Effect of phosphorothioate substitutions on DNA cleavage by Escherichia coli DNA topoisomerase I

To evaluate the structural influence of the DNA phosphate backbone on the activity of Escherichia coli DNA topoisomerase I, modified forms of oligonucleotide dA(7) were synthesized with a chiral phosphorothioate replacing the non-bridging oxygens at each position along the backbone. A deoxy-iodo-uracil replaced the 5'-base to crosslink the oligonucleotides by ultraviolet (UV) and assess binding affinity. At the scissile phosphate there was little effect on the cleavage rate. At the +1 phosphate, the rectus phosphorus (Rp)-thio-substitution reduced the rate of cleavage by a factor of 10. At the +3 and -2 positions from the scissile bond, the Rp-isomer was cleaved at a faster rate than the sinister phosphorus (Sp)-isomer. The results demonstrate the importance of backbone contacts between DNA substrate and E. coli topoisomerase I.     

Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes.

The chemical synthesis is described of oligoribonucleotides containing a single phosphorothioate linkage of defined Rp and Sp configuration. The oligoribonucleotides were used as substrates in the study of the mechanism of cleavage of an RNA hammerhead domain having the phosphorothioate group at the cleavage site. Whereas the Rp isomer was cleaved only very slowly in the presence of magnesium ion, the rate of cleavage of the Sp isomer was only slightly reduced from that of the unmodified phosphodiester. This finding gives further evidence for the hypothesis that the magnesium ion is bound to the pro-R oxygen in the transition state of the hammerhead cleavage reaction. Also, inversion of configuration at phosphorus is confirmed for a two-stranded hammerhead.