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1.
Bacteriophage phi X174 encoded gene A protein is an enzyme required for initiation and termination of successive rounds of rolling circle phi X DNA replication. This enzyme catalyses cleavage and ligation of a phosphodiester bond between nucleotide residues G and A at the phi X origin. The cleavage reaction which occurs during initiation involves formation of a free GOH residue at one end and a covalent bond between tyrosine-OH of the gene A protein and 5' phosphate of the A residue, at the other end of the cleavage site. During termination the covalently bound gene A protein cleaves the phosphodiester bond between G and A at the regenerated origin and ligates the 3' and 5' ends of the displaced genome-length viral DNA to form a circle. Since tyrosyl-OH mediated rearrangements of phosphodiester bonds in DNA may also apply to other enzymes involved in replication or recombination such as topoisomerases we have studied this interesting mechanism in greater detail. Analysis of 32P-labelled gene A protein-DNA complex by tryptic digestion followed by sequencing of 32P-containing peptides showed that two tyrosyl residues in the repeating sequence tyr-val-ala-lys-tyr-val-asn-lys participate in phosphodiester bond cleavage. Either one of these tyrosyl residues can function as the acceptor of the DNA chain. In an alpha-helix the side chains of these tyrosyl residues are in juxtaposition. An enzymatic mechanism is proposed in which these two tyrosyl-OH groups participate in an alternating manner in successive cleavage and ligations which occur during phosphodiester bond rearrangements of DNA.  相似文献   

2.
Cleavage of DNA by mammalian DNA topoisomerase II   总被引:46,自引:0,他引:46  
Using the P4 unknotting assay, DNA topoisomerase II has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase II can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerase II-DNA complex and is reversible with respect to salt and temperature. The product after reversal of the cleavage reaction remains supertwisted, suggesting that the two ends of the putatively broken DNA are held tightly by the topoisomerase. Alternatively, the enzyme-DNA interaction is noncovalent, and the covalent linking of topoisomerase to DNA is induced by the protein denaturant. Detailed characterization of the cleavage products has revealed that topoisomerase II cuts DNA with a four-base stagger and is covalently linked to the protruding 5'-phosphoryl ends of each broken DNA strand. Calf thymus DNA topoisomerase II cuts SV40 DNA at multiple and specific sites. However, no sequence homology has been found among the cleavage sites as determined by direct nucleotide-sequencing studies.  相似文献   

3.
Escherichia coli DNA topoisomerase I catalyzes relaxation of negatively supercoiled DNA. The reaction proceeds through a covalent intermediate, the cleavable complex, in which the DNA is cleaved and the enzyme is linked to the DNA via a phosphotyrosine linkage. Each molecule of E. coli DNA topoisomerase I has been shown to have three tightly bound zinc(II) ions required for relaxation activity (Tse-Dinh, Y.-C., and Beran-Steed, R.K. (1988) J. Biol. Chem. 263, 15857-15859). It is shown here that Cd(II) could replace Zn(II) in reconstitution of active enzyme from apoprotein. The role of metal was analyzed by studying the partial reactions. The apoenzyme was deficient in sodium dodecyl sulfate-induced cleavage of supercoiled PM2 phage DNA. Formation of covalent complex with linear single-stranded DNA was also reduced in the absence of metal. However, the cleavage of small oligonucleotide was not affected, and the apoenzyme could religate the covalently bound oligonucleotide to another DNA molecule. Assay of noncovalent complex formation by retention of 5'-labeled DNA on filters showed that the apoenzyme was not inhibited in noncovalent binding to DNA. It is proposed that zinc(II) coordination in E. coli DNA topoisomerase I is required for the transition of the noncovalent complex with DNA to the cleavable state.  相似文献   

4.
We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes. Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA. The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a property similar to the eukaryotic type I topoisomerases. The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.  相似文献   

5.
Eukaryotic type 1B topoisomerases act by forming covalent enzyme-DNA intermediates that transiently nick DNA and thereby release DNA supercoils. Here we present a study of the topoisomerase encoded by the pathogenic poxvirus molluscum contagiosum. Our studies of DNA sites favored for catalysis reveal a larger recognition site than the 5'-(T/C)CCTT-3' sequence previously identified for poxvirus topoisomerases. Separate assays of initial DNA binding and covalent complex formation revealed that different DNA sequences were important for each reaction step. The location of the protein-DNA contacts was mapped by analyzing mutant sites and inosine-substituted DNAs. Some of the bases flanking the 5'-(T/C)CCTT-3' sequence were selectively important for covalent complex formation but not initial DNA binding. Interactions important for catalysis were probed with 5'-bridging phosphorothiolates at the site of strand cleavage, which permitted covalent complex formation but prevented subsequent religation. Kinetic studies revealed that the flanking sequences that promoted recovery of covalent complexes increased initial cleavage instead of inhibiting resealing of the nicked intermediate. These data 1) indicate that previously unidentified DNA contacts can accelerate a step between initial binding and covalent complex formation and 2) help specify models for conformational changes promoting catalysis.  相似文献   

6.
A method has been used to quantitate the reaction between eukaryotic type I DNA topoisomerase and topological forms of DNA. This procedure (Trask, D.K., DiDonato, J.D. and Muller, M.T. (1984) Eur. Mol. Biol. Organ. J. 3, 671-676) measures the efficiency of DNA cleavage and concurrent formation of a covalent enzyme/DNA complex. Eukaryotic type I topoisomerases react preferentially by 5-10-fold with supercoiled DNA. The effect of supercoiling is clearly evident in that both the initial rate and final extent of the reaction is elevated. Because the dissociation rate is much lower than the association rate, it is possible to isolate native topoisomerase/DNA complexes. These complexes are comprised of enzyme molecules which are catalytically active when challenged with a second supercoiled DNA substrate. Collectively, the data support the conclusion that a functional intermediate in the reaction sequence is being detected and that the avian topoisomerase I preferentially cleaves supercoiled DNA.  相似文献   

7.
The phi X174 A protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated end. To determine the nature of the covalent linkage and the amino acid involved, we used the A protein to cleave DNA synthesized in vitro with [alpha-32P]dATP to form the complex of A protein covalently linked to single-stranded DNA. The complex was then digested with DNase I, and the 32P-labeled A protein was isolated by electrophoresis on polyacrylamide gels. The isolated complex was treated extensively with trypsin, and the released peptide-oligonucleotide complexes were incubated with formic acid and diphenylamine (Burton reaction). The Burton reaction caused a transfer of the labeled phosphate from dAMP to the peptide. The labeled phosphopeptides were isolated and hydrolyzed, revealing a linkage of the phosphate to a tyrosine. These results indicate that the A protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated terminus by a tyrosyl-dAMP phosphodiester bond.  相似文献   

8.
The TOP3 gene of the yeast Saccharomyces cerevisiae was postulated to encode a DNA topoisomerase, based on its sequence homology to Escherichia coli DNA topoisomerase I and the suppression of the poor growth phenotype of top3 mutants by the expression of the E. coli enzyme (Wallis, J.W., Chrebet, G., Brodsky, G., Golfe, M., and Rothstein, R. (1989) Cell 58, 409-419). We have purified the yeast TOP3 gene product to near homogeneity as a 74-kDA protein from yeast cells lacking DNA topoisomerase I and overexpressing a plasmid-borne TOP3 gene linked to a phosphate-regulated yeast PHO5 gene promoter. The purified protein possesses a distinct DNA topoisomerase activity: similar to E. coli DNA topoisomerases I and III, it partially relaxes negatively but not positively supercoiled DNA. Several experiments, including the use of a negatively supercoiled heteroduplex DNA containing a 29-nucleotide single-stranded loop, indicate that the activity has a strong preference for single-stranded DNA. A protein-DNA covalent complex in which the 74-kDa protein is linked to a 5' DNA phosphoryl group has been identified, and the nucleotide sequences of 30 sites of DNA-protein covalent complex formation have been determined. These sequences differ from those recognized by E. coli DNA topoisomerase I but resemble those recognized by E. coli DNA topoisomerase III. Based on these results, the yeast TOP3 gene product can formally be termed S. cerevisiae DNA topoisomerase III. Analysis of supercoiling of intracellular yeast plasmids in various DNA topoisomerase mutants indicates that yeast DNA topoisomerase III has at most a weak activity in relaxing negatively supercoiled double-stranded DNA in vivo, in accordance with the characteristics of the purified enzyme.  相似文献   

9.
ResT is a member of the telomere resolvases, a newly discovered class of DNA breakage and reunion enzymes. These enzymes are involved in the formation of co-valently closed hairpin DNA ends that are found in linear prokaryotic chromosomes and plasmids. The hairpins are generated by telomere resolution, where the replicated linear DNA ends are processed by DNA breakage followed by joining of DNA free ends to the complementary strand of the same molecule. Previous studies have shown that ResT catalyzes hairpin formation through a two-step transesterification similar to tyrosine recombinases and type IB topoisomerases. In the present study we have probed the reaction mechanism of ResT. The enzyme was found to efficiently utilize a substrate with a 5'-bridging phosphorothiolate at each cleavage site, similar to tyrosine recombinases/type IB topoisomerases. Using such a substrate to trap the covalent protein-DNA intermediate, coupled with affinity purification and mass spectroscopy, we report a new, non-radioactive approach to directly determine the position of the amino acid in the protein, which is linked to the DNA. We report that tyrosine 335 is the active site nucleophile in ResT, strengthening the link between ResT and tyrosine recombinases/type IB topoisomerases. However, a distinct pattern of catalytic residues with similarities, but distinct differences from the above enzymes was suggested. The differences include the apparent absence of a general acid catalyst, as well as the dispensability of the final histidine in the RKHRHY hexad. Finally, two signature motifs (GRR(2X)E(6X)F and LGH(4-6X)T(3X)Y) near the catalytic residues of aligned telomere resolvases are noted.  相似文献   

10.
Type IB topoisomerases cleave and rejoin DNA strands through a stable covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate. The stability of the intermediate is a two-edged sword; it preserves genome integrity during supercoil relaxation, but it also reinforces the toxicity of drugs and lesions that interfere with the DNA rejoining step. Here, we identify a key determinant of the stability of the complex by showing that introduction of an Sp or Rp methylphosphonate linkage at the cleavage site transforms topoisomerase IB into a potent endonuclease. The nuclease reaction entails formation and surprisingly rapid hydrolysis of a covalent enzyme-DNA methylphosphonate intermediate. The approximately 30,000-fold acceleration in the rate of hydrolysis of a methylphosphonate versus phosphodiester suggests that repulsion of water by the DNA phosphate anion suppresses the latent nuclease function of topoisomerase IB. These findings expose an Achilles' heel of topoisomerases as guardians of the genome, and they have broad implications for understanding enzymatic phosphoryl transfer.  相似文献   

11.
Vaccinia topoisomerase forms a covalent protein-DNA intermediate at 5'-CCCTT downward arrow sites in duplex DNA. The T downward arrow nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that mutant enzymes containing glutamate, cysteine or histidine in lieu of Tyr-274 catalyze endonucleolytic cleavage of a 60 bp duplex DNA at the CCCTT downward arrow site to yield a 3' phosphate-terminated product. The Cys-274 mutant forms trace levels of a covalent protein-DNA complex, suggesting that the DNA cleavage reaction may proceed through a cysteinyl-phosphate intermediate. However, the His-274 and Glu-274 mutants evince no detectable accumulation of a covalent protein-DNA adduct. Glu-274 is the most active of the mutants tested. The pH dependence of the endonuclease activity of Glu-274 (optimum pH = 6.5) is distinct from that of the wild-type enzyme in hydrolysis of the covalent adduct (optimum pH = 9.5). At pH 6.5, the Glu-274 endonuclease reaction is slower by 5-6 orders of magnitude than the rate of covalent adduct formation by the wild-type topoisomerase, but is approximately 20 times faster than the rate of hydrolysis by the wild-type covalent adduct. We discuss two potential mechanisms to account for the apparent conversion of a topoisomerase into an endonuclease.  相似文献   

12.
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.  相似文献   

13.
Mizuuchi K  Nobbs TJ  Halford SE  Adzuma K  Qin J 《Biochemistry》1999,38(14):4640-4648
A new method was developed for tracking the stereochemical path of enzymatic cleavage of DNA. DNA with a phosphorothioate of known chirality at the scissile bond is cleaved by the enzyme in H218O. The cleavage produces a DNA molecule with the 5'-[16O,18O, S]-thiophosphoryl group, whose chirality depends on whether the cleavage reaction proceeds by a single-step hydrolysis mechanism or by a two-step mechanism involving a protein-DNA covalent intermediate. To determine this chirality, the cleaved DNA is joined to an oligonucleotide by DNA ligase. Given the strict stereochemistry of the DNA ligase reaction, determined here, the original chirality of the phosphorothioate dictates whether the 18O is retained or lost in the ligation product, which can be determined by mass spectrometry. This method has advantages over previous methods in that it is not restricted to particular DNA sequences, requires substantially less material, and avoids purification of the products at intermediate stages in the procedure. The method was validated by confirming that DNA cleavage by the EcoRI restriction endonuclease causes inversion of configuration at the scissile phosphate. It was then applied to the reactions of the SfiI and HpaII endonucleases and the MuA transposase. In all three cases, DNA cleavage proceeded with inversion of configuration, indicating direct hydrolysis of the phosphodiester bond by water as opposed to a reaction involving a covalent enzyme-DNA intermediate.  相似文献   

14.
DNA topoisomerases play essential roles in many DNA metabolic processes. It has been suggested that topoisomerases play an essential role in DNA repair. Topoisomerases can introduce DNA damage upon exposure to drugs that stabilize the covalent protein-DNA intermediate of the topoisomerase reaction. Lesions in DNA are also able to trap topoisomerase-DNA intermediates, suggesting that topoisomerases have the potential to either assist in DNA repair by locating sites of damage or exacerbating DNA damage by generation of additional damage at the site of a lesion. We have shown that overexpression of yeast topoisomerase I (TOP1) conferred hypersensitivity to methyl methanesulfonate and other DNA-damaging agents, whereas expression of a catalytically inactive enzyme did not. Overexpression of topoisomerase II did not change the sensitivity of cells to these DNA-damaging agents. Yeast cells lacking TOP1 were not more resistant to DNA damage than cells expressing wild type levels of the enzyme. Yeast topoisomerase I covalent complexes can be trapped efficiently on UV-damaged DNA. We suggest that TOP1 does not participate in the repair of DNA damage in yeast cells. However, the enzyme has the potential of exacerbating DNA damage by forming covalent DNA-protein complexes at sites of DNA damage.  相似文献   

15.
Type IA DNA topoisomerases, typically found in bacteria, are essential enzymes that catalyse the DNA relaxation of negative supercoils. DNA gyrase is the only type II topoisomerase that can carry out the opposite reaction (i.e. the introduction of the DNA supercoils). A number of diverse molecules target DNA gyrase. However, inhibitors that arrest the activity of bacterial topoisomerase I at low concentrations remain to be identified. Towards this end, as a proof of principle, monoclonal antibodies that inhibit Mycobacterium smegmatis topoisomerase I have been characterized and the specific inhibition of Mycobacterium smegmatis topoisomerase I by a monoclonal antibody, 2F3G4, at a nanomolar concentration is described. The enzyme-bound monoclonal antibody stimulated the first transesterification reaction leading to enhanced DNA cleavage, without significantly altering the religation activity of the enzyme. The stimulated DNA cleavage resulted in perturbation of the cleavage-religation equilibrium, increasing single-strand nicks and protein-DNA covalent adducts. Monoclonal antibodies with such a mechanism of inhibition can serve as invaluable tools for probing the structure and mechanism of the enzyme, as well as in the design of novel inhibitors that arrest enzyme activity.  相似文献   

16.
Y Yamashita  S Kawada  N Fujii  H Nakano 《Biochemistry》1991,30(24):5838-5845
Saintopin is an antitumor antibiotic recently discovered in mechanistically oriented screening using purified calf thymus DNA topoisomerases. Saintopin induced topoisomerase I mediated DNA cleavage comparable to that of camptothecin, and topoisomerase II mediated DNA cleavage equipotent to those of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) or 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16). Treatment of a reaction mixture containing saintopin and topoisomerase I or II with either elevated temperature (65 degrees C) or higher salt concentration (0.5 M NaCl) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I and II mediated DNA cleavage induced by saintopin is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex". Consistent with the cleavable complex formation with both topoisomerases, saintopin inhibited catalytic activities of both topoisomerase I and topoisomerase II. The DNA cleavage intensity pattern induced by saintopin with topoisomerase I was different from that by camptothecin. A difference in cleavage pattern was also detected between saintopin and m-AMSA or VP-16 in topoisomerase II mediated DNA cleavage. DNA unwinding assay using T4 DNA ligase showed that saintopin is a weak DNA intercalator like m-AMSA. Thus, saintopin represents a new class of antitumor agent that can induce both mammalian DNA topoisomerase I and mammalian DNA topisomerase II mediated DNA cleavage.  相似文献   

17.
Topoisomerases form a covalent enzyme-DNA intermediate after initial DNA cleavage. Trapping of the cleavage complex formed by type IIA topoisomerases initiates the bactericidal action of fluoroquinolones. It should be possible also to identify novel antibacterial lead compounds that act with a similar mechanism on type IA bacterial topoisomerases. The cellular response and repair pathways for trapped topoisomerase complexes remain to be fully elucidated. The RuvAB and RecG proteins could play a role in the conversion of the initial protein-DNA complex to double-strand breaks and also in the resolution of the Holliday junction during homologous recombination. Escherichia coli strains with ruvA and recG mutations are found to have increased sensitivity to low levels of norfloxacin treatment, but the mutations had more pronounced effects on survival following the accumulation of covalent complexes formed by mutant topoisomerase I defective in DNA religation. Covalent topoisomerase I and DNA gyrase complexes are converted into double-strand breaks for SOS induction by the RecBCD pathway. SOS induction following topoisomerase I complex accumulation is significantly lower in the ruvA and recG mutants than in the wild-type background, suggesting that RuvAB and RecG may play a role in converting the initial single-strand DNA-protein cleavage complex into a double-strand break prior to repair by homologous recombination. The use of a ruvB mutant proficient in homologous recombination but not in replication fork reversal demonstrated that the replication fork reversal function of RuvAB is required for SOS induction by the covalent complex formed by topoisomerase I.DNA topoisomerases can modulate DNA superhelicity and help overcome topological barriers in cellular processes by cleaving the DNA backbone phosphodiester linkage to allow topological changes in DNA substrates. The ends of the cleaved DNA are covalently linked to an active-site tyrosine on the topoisomerase proteins in cleavage complex intermediates. Covalent protein-DNA complexes exist only transiently during catalysis because the cleaved DNA is rapidly religated. The stabilization of covalent complexes formed by human topoisomerase I or II due to the action of certain anticancer drugs results in the apoptotic death of cancer cells. Quinolone antibiotics are highly bactericidal because they cause the accumulation of covalent complexes formed by bacterial DNA gyrase and topoisomerase IV enzymes. Although a similar topoisomerase poison inhibitor remains to be identified for bacterial type IA topoisomerases, bacterial topoisomerase I complex accumulation due to mutations that inhibit DNA religation has also been shown to cause rapid bacterial cell death (4, 36). The requirement of a DNA cleavage step in the mechanism of action of topoisomerases increases the vulnerability of cells to conditions that would trap the covalent protein-DNA complex. These conditions include the presence of DNA intercalators, toxic metabolites, and DNA lesions, as well as protein thiolation (9, 28-31, 38). Response to and repair of the trapped covalent topoisomerase-DNA complex are thus needed for cell survival. In eukaryotes, 3′-tyrosyl DNA phosphodiesterase (TDP1) and 5′-tyrosyl DNA phosphodiesterase (TDP2), which can cleave the covalent linkage between topoisomerases and DNA, have been identified (8, 15, 27). Tyrosyl DNA phosphodiesterases have not been identified in bacteria. Repair of covalent bacterial topoisomerase-DNA complexes may require the action of endonucleases to remove the DNA-bound topoisomerase proteins, similar to the Rad1-Rad10 repair pathway characterized in yeast (37). In Escherichia coli, covalent topoisomerase I and DNA gyrase complexes have been shown to be processed into double-strand DNA breaks (DSB), which are then repaired via the RecBCD-mediated RecA homologous recombination pathway with induction of the SOS regulon (24, 34). The RuvABC and RecG activities could play significant roles in the response to the covalent topoisomerase complexes. They are both capable of resolving the Holliday junctions following DSB formation in the later stages of homologous recombination repair (11). SbcCD has been shown previously to remove protein from a protein-bound DNA end with nucleolytic activity to create a DSB (7). In addition, it is also possible that RuvAB and RecG might act at arrested forks to process replication forks blocked by the covalently bound topoisomerase proteins and generate DSB substrates for RecBCD (1, 32). Previous studies have not clearly elucidated the roles of RuvABC and RecG in the response to covalent topoisomerase complexes. We examine here the effects of mutations in the ruvA and recG genes on both bacterial survival and SOS induction following the accumulation of covalent topoisomerase I or gyrase complexes with cleaved DNA.  相似文献   

18.
Crithidia fasciculata nicking enzyme (Shlomai, J., and Linial, M. (1986) J. Biol. Chem. 261, 16219-16225) interrupts a single phosphodiester bond in duplex DNA circles from various sources, only in their supercoiled form, but not following their relaxation by DNA topoisomerases. However, this requirement for DNA substrate supercoiling was not observed using the natural kinetoplast DNA as a substrate. Relaxed kinetoplast DNA minicircles, either free or topologically linked, were efficiently nicked by the enzyme. Furthermore, bacterial plasmids, containing a unit length kinetoplast DNA minicircle insert, were used as substrates for nicking in their relaxed form. This capacity to activate a relaxed DNA topoisomer as a substrate for nicking is an intrinsic property of the sequence-directed bend, naturally present in kinetoplast DNA. The 211-base pair fragment of the bent region from C. fasciculata kinetoplast DNA could support the nicking of a relaxed DNA substrate in a reaction dependent upon the DNA helix curvature.  相似文献   

19.
To probe the mechanism of the reversible DNA phosphodiester bond cleavage and religation mechanism of the type I topoisomerase from vaccinia virus, we have synthesized DNA substrates carrying a single nonbridging Rp- or Sp-phosphorothioate (Ps) modification at the scissile phosphodiester (Pd) bond. Analysis of the stereochemical outcome of the net cleavage and rejoining reaction established that the reaction proceeds with retention of configuration, as expected for a double-displacement mechanism. Single-turnover kinetic studies on irreversible strand cleavage using 18/24 mer suicide substrates showed thio effects (k(Pd)/k(Ps)) of 340- and 30-fold for the Rp-Ps and Sp-Ps stereoisomers, respectively, but approximately 10-fold smaller thio effects for the reverse single-turnover religation reaction (Rp-Ps = 30 and Sp-Ps = 3). As compared to the smaller suicide cleavage substrates, approach-to-equilibrium cleavage studies using 32/32 mer substrates showed 7-9-fold smaller thio effects on cleavage, similar effects on religation, and the same ratio of the Rp to Sp thio effect as the suicide cleavage reaction ( approximately 10). In general, thio effects of 2.4-7.2-fold on the cleavage equilibrium are observed for the wild-type and H265A enzymes, suggesting differences in the interactions of the enzyme with the nonbridging sulfur in the noncovalent and covalent complexes. Studies of the cleavage, religation, and approach-to-equilibrium reactions catalyzed by the H265A active site mutant revealed a stereoselective, 11-fold decrease in the Rp-thio effect on cleavage and religation as compared to the wild-type enzyme. This result suggests that His-265 interacts with the nonbridging pro-Rp oxygen in the transition state for cleavage and religation, consistent with the arrangement of this conserved residue in the crystal structure of the human topoisomerase-DNA complex. In general, the greatest effect of thio substitution and the H265A mutation is to destabilize the transition state, with smaller effects on substrate binding. The interaction of His-265 with the pro-Rp nonbridging oxygen is inconsistent with the proposal that this conserved residue acts as a general acid in the strand cleavage reaction.  相似文献   

20.
DNA helicase I, encoded on the Escherichia coli F plasmid, catalyzes a site- and strand-specific nicking reaction within the F plasmid origin of transfer (oriT) to initiate conjugative DNA strand transfer. The product of the nicking reaction contains a single phosphodiester bond interruption as determined by single-nucleotide resolution mapping of both sides of the nick site. This analysis has demonstrated that the nick is located at precisely the same site previously shown to be nicked in vivo (T. L. Thompson, M. B. Centola, and R. C. Deonier, J. Mol. Biol. 207:505-512, 1989). In addition, studies with two oriT point mutants have confirmed the specificity of the in vitro reaction. Characterization of the nicked DNA product has revealed a modified 5' end and a 3' OH available for extension by E. coli DNA polymerase I. Precipitation of nicked DNA with cold KCl in the presence of sodium dodecyl sulfate suggests the existence of protein covalently attached to the nicked DNA molecule. The covalent nature of this interaction has been directly demonstrated by transfer of radiolabeled phosphate from DNA to protein. On the basis of these results, we propose that helicase I becomes covalently bound to the 5' end of the nicked DNA strand as part of the reaction mechanism for phosphodiester bond cleavage. A model is presented to suggest how helicase I could nick the F plasmid at oriT and subsequently unwind the duplex DNA to provide single-stranded DNA for strand transfer during bacterial conjugation.  相似文献   

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