Open complex formation around a lesion during nucleotide excision repair provides a structure for cleavage by human XPG protein.

Human XPG nuclease makes the 3' incision during nucleotide excision repair of DNA. The enzyme cleaves model DNA bubble structures specifically near the junction of unpaired DNA with a duplex region. It is not yet known, however, whether an unpaired structure is an intermediate during actual DNA repair. We find here that XPG requires opening of >5 bp for efficient cleavage. To seek direct evidence for formation of an open structure around a lesion in DNA during a nucleotide excision repair reaction in vitro, KMnO4 footprinting experiments were performed on a damaged DNA molecule bearing a uniquely placed cisplatin adduct. An unwound open complex spanning approximately 25 nucleotides was observed that extended to the positions of 5' and 3' incision sites and was dependent on XPA protein and on ATP. Opening during repair occurred prior to strand incision by XPG.     

DNA-cleavage studies on N-substituted monocyclic enediynes: enhancement of potency by incorporation of intercalating or electron poor aromatic ring and subsequent design of a novel phototriggerable acyclic enediyne

A number of novel N-substituted enediynes (azaenediynes) 1-4 were synthesized as DNA cleaving agents. Enhancement of DNA cleavage potency was observed with those compounds which could interact with DNA through intercalation of the extended aromatic ring or through electrostatic attraction with electron poor aromatic ring. An acyclic enediyne 5 with a novel phototriggerable device was also synthesized and its DNA-cleaving activity was established.     

Benzo[c]phenanthrene adducts and nogalamycin inhibit DNA transesterification by vaccinia topoisomerase

Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a specific target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow N(-1) in duplex DNA. Here we study the effects of position-specific DNA intercalators on the rate and extent of single-turnover DNA transesterification. Chiral C-1 R and S trans-opened 3,4-diol 1,2-epoxide adducts of benzo[c]phenanthrene (BcPh) were introduced at single N2-deoxyguanosine and N6-deoxyadenosine positions within the 3'-G(+5)G(+4)G(+3)A(+2)A(+1)T(-1)A(-2) sequence of the nonscissile DNA strand. Transesterification was unaffected by BcPh intercalation between the +6 and +5 base pairs, slowed 4-fold by intercalation between the +5 and +4 base pairs, and virtually abolished by BcPh intercalation between the +4 and +3 base pairs and the +3 and +2 base pairs. Intercalation between the +2 and +1 base pairs by the +2R BcPh dA adduct abolished transesterification, whereas the overlapping +1S BcPh dA adduct slowed the rate of transesterification by a factor of 2700, with little effect upon the extent of the reaction. Intercalation at the scissile phosphodiester (between the +1 and -1 base pairs) slowed transesterification by a factor of 450. BcPh intercalation between the -1 and -2 base pairs slowed cleavage by two orders of magnitude, but intercalation between the -2 and -3 base pairs had little effect. The anthracycline drug nogalamycin, a non-covalent intercalator with preference for 5'-TG dinucleotides, inhibited the single-turnover DNA cleavage reaction of vaccinia topoisomerase with an IC50 of 0.7 microM. Nogalamycin was most effective when the drug was pre-incubated with DNA and when the cleavage target site was 5'-CCCTT/G instead of 5'-CCCTT/A. These findings demarcate upstream and downstream boundaries of the functional interface of vaccinia topoisomerase with its DNA target site.     

Theoretical study of the catalytic reaction mechanism of MndD

Manganese-dependent homoprotocatechuate 2,3-dioxygenase (MndD) is an enzyme taking part in the catabolism of aromatic compounds in the environment. It uses molecular oxygen to perform an extradiol cleavage of the ring of the ortho-dihydroxylated aromatic compound homoprotocatechuate. A theoretical investigation of the reaction path for MndD was performed using hybrid density functional theory with the B3LYP functional, and a catalytic mechanism has been suggested. Models of different size were built from the crystal structure of the enzyme and were used in the search for intermediates and transition states. It was found that the substrate first binds at the active site as a monoanion. Next the dioxygen is bound, forming a hydroperoxo intermediate. The O–O bond, activated in this way undergoes homolytic cleavage leading to an oxyl and then to an extra epoxide radical with subsequent opening of the aromatic ring. The lactone ring is then hydrolyzed by the Mn-bound OH group, and the final product is obtained in the last reaction steps. Alternative reaction paths were considered, and their calculated barriers were found to be higher than for the suggested mechanism. The selectivity between the extra- and intra-cleavage pathways was found to be determined by the barriers for the decay of the radical state.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Kinetic and mechanistic investigation of the selective acidolysis of the C-terminal amide bond of N-acyl-N,alpha,alpha-trialkyl glycine amides.

Accurate rate constants were calculated from HPLC kinetic measurements of the selective acidolysis of the C-terminal amide bond of eight N-acyl-N-(4-methoxybenzyl)-alpha,alpha-trialkyl glycine amides in TFA at 25.00 degrees C. The results were in all cases consistent with a first order behaviour with respect to the substrate and, apparently, also to the acid, and a clear relationship between reactivity and structure could be observed. The data collected also allowed experimental evidence to be obtained for the first time in support of the previously postulated formation of an intermediate oxazolonium salt. In the case of the more crowded species this intermediate compound undergoes slow hydrolytic ring opening, which takes place in competition with cleavage of the N-alkyl group to give another oxazolonium derivative that hydrolysed still more slowly. The stability of the intermediate cyclic compounds may result either from conjugation of the phenyl group with the oxazolonium ring in the case of N-benzoyl derivatives, or from conformational assistance imparted by the bulky amino acid side chains of the alpha,alpha-dialkyl glycine species, or both. The loss of the N-alkyl group also seems to be assisted by the bulkiness of the amino acid side chains, which thus tends to decrease the selectivity of cleavage.     

The role of side chains in the interaction of new antitumor pyrimidoacridinetriones with DNA: molecular dynamics simulations

Pyrimidoacridinetriones (PATs) are a new group of highly active antitumor compounds. It seems reasonable to assume that, like for some other acridine derivatives, intercalation into DNA is a necessary, however not a sufficient condition for antitumor activity of these compounds. Rational design of new compounds of this chemotype requires knowledge about the structure of the intercalation complex, as well as about interactions responsible for its stability. Computer simulation techniques such as molecular dynamics (MD) may provide valuable information about these problems. The results of MD simulations performed for three rationally selected PATs are presented in this paper. The compounds differ in the number and position of side chains. Each of the compounds was simulated in two systems: i) in water, and ii) in the intercalation complex with the dodecamer duplex d(GCGCGCGCGCGC)2. The orientation of the side chain in relation to the ring system is determined by the position of its attachment. Orientation of the ring system inside the intercalation cavity depends on the number and position of side chain(s). The conformations of the side chain(s) of all PATs studied in the intercalation complex were found to be very similar to those observed in water.     

Radical rebound mechanism in cytochrome P-450-catalyzed hydroxylation of the multifaceted radical clocks alpha- and beta-thujone

Alpha-thujone (1alpha) and beta-thujone (1beta) were used to investigate the mechanism of hydrocarbon hydroxylation by cytochromes P-450(cam) (CYP101) and P-450(BM3) (CYP102). The thujones are hydroxylated by these enzymes at various positions, but oxidation at C-4 gives rise to both rearranged and unrearranged hydroxylation products. Rearranged products result from the formation of a radical intermediate that can undergo either inversion of stereochemistry or ring opening of the adjacent cyclopropane ring. Both of these rearrangements, as well as a C-4 desaturation reaction, are observed. The ring opening clock gives oxygen rebound rates that range from 0.2 x 10(10) to 2.8 x 10(10) s(-1) for the different substrate and enzyme combinations. The C-4 inversion reaction provides independent confirmation of a radical intermediate. The phenol product expected if a C-4 cationic rather than radical intermediate is formed is not detected. The results are consistent with a two-state process and provide support for a radical rebound but not a hydroperoxide insertion mechanism for cytochrome P-450 hydroxylation.     

Structural analysis of Pseudomonas 1-aminocyclopropane-1-carboxylate deaminase complexes: insight into the mechanism of a unique pyridoxal-5'-phosphate dependent cyclopropane ring-opening reaction

1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a pyridoxal 5'-phosphate (PLP) dependent enzyme catalyzing the opening of the cyclopropane ring of ACC to give alpha-ketobutyric acid and ammonia as the products. This ring cleavage reaction is unusual because the substrate, ACC, contains no abstractable alpha-proton and the carboxyl group is retained in the product. How the reaction is initiated to generate an alpha-carbanionic intermediate, which is the common entry for most PLP-dependent reactions, is not obvious. To gain insight into this unusual ring-opening reaction, we have solved the crystal structures of ACC deaminase from Pseudomonas sp. ACP in complex with substrate ACC, an inhibitor, 1-aminocyclopropane-1-phosphonate (ACP), the product alpha-ketobutyrate, and two d-amino acids. Several notable observations of these structural studies include the following: (1) a typically elusive gem-diamine intermediate is trapped in the enzyme complex with ACC or ACP; (2) Tyr294 is in close proximity (3.0 A) to the pro-S methylene carbon of ACC in the gem-diamine complexes, implicating a direct role of this residue in the ring-opening reaction; (3) Tyr294 may also be responsible for the abstraction of the alpha-proton from d-amino acids, a prelude to the subsequent deamination reaction; (4) the steric hindrance precludes accessibility of active site functional groups to the l-amino acid substrates and may account for the stereospecificity of this enzyme toward d-amino acids. These structural data provide evidence favoring a mechanism in which the ring cleavage is induced by a nucleophilic attack at the pro-S beta-methylene carbon of ACC, with Tyr294 as the nucleophile. However, these observations are also consistent with an alternative mechanistic possibility in which the ring opening is acid-catalyzed and may be facilitated by charge relay through PLP, where Tyr294 functions as a general acid. The results of mutagenesis studies corroborated the assigned critical role for Tyr294 in the catalysis.     

Formation of modified cleavage termini from the reaction of chromium(V) with DNA

Reaction of a 25 bp oligonucleotide with the high valent chromium complex, bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) (Cr(V)-EHBA) produced both Frank- and alkali-labile strand breaks that were sequence-neutral. Frank strand break formation was found to be O2-dependent while formation of alkali-labile strand breaks were O2-independent. Reaction of Cr(V)-EHBA with the 5'-32P-labeled oligomer under oxygenated conditions formed the modified 3'-terminus, 3'-phosphoglycolate, as well as the 3'-phosphate terminus. Formation of the 3'-phosphoglycolate termini, and the O2 dependence of the reactions were consistent with a mechanism involving abstraction of the C4' hydrogen atom from the deoxyribose moiety of DNA. Identical reactions using the 3'-32P-labeled oligomer yielded only 5'-phosphate termini as assigned by co-migration with Maxam-Gilbert markers. Analogous cleavage profiles and modified termini were observed for the reaction of Cr(V)-EHBA and DNA in the presence of hydrogen peroxide. With the addition of hydrogen peroxide, the DNA cleavage reactions were O2-independent and the level of DNA cleavage was enhanced over that observed with Cr(V)-EHBA alone. These findings suggest an oxidation mechanism whereby a reductive intermediate of the carcinogen chromate, Cr(V), can cause DNA damage that mimics oxygen radical DNA damaging pathways.     

Racemization of chlorthalidone in the presence of liposomes

The extent of racemization of (+)-chlorthalidone as a function of pH is examined. The minimum of the log K/pH curve is pH 3. The reaction mechanism of inversion is postulated to involve a carbenium cation over the entire pH range and a ring opening reaction in the alkaline range. The influence of liposomes on the inversion rate is also studied, retardation of the racemization rate being observed with increasing liposome concentration. A model of drug distribution between liposome phase and aqueous phase based on the Nernst distribution principle is presented and reaction kinetic aspects are considered. © 1993 Wiley-Liss, Inc.     

The reaction mechanism of DNA glycosylase/AP lyases at abasic sites

DNA glycosylase and glycosylase/abasic (AP) lyases are the enzymes responsible for initiating the base excision repair pathway by recognizing the damaged target base and catalyzing the breakage of the base-sugar glycosyl bond. The subset of glycosylases that have an associated AP lyase activity also catalyze DNA strand breakage at the resulting or preexisting AP site via a beta-elimination reaction, proceeding from an enzyme-DNA imino intermediate. Two distinct mechanisms have been proposed for the formation of this intermediate. These mechanisms essentially differ in the nature of the first bond broken and the timing of the opening of the deoxyribose ring. The data presented here demonstrate that the combined rate of sugar ring opening and reduction of the sugar is significantly slower than the rate of formation of a T4-pyrimidine dimer glycosylase (T4-pdg)-DNA intermediate. Using a methyl-deoxyribofuranose AP-site analogue that is incapable of undergoing sugar ring opening, it was demonstrated that the T4-pdg reaction can initiate at the ring-closed form, albeit at a drastically reduced rate. T4-pdg preferentially cleaved the beta-anomer of the methyl-deoxyribofuranose AP site analogue. This is consistent with a mechanism in which the methoxy group is backside-displaced by the amino group from the alpha-face of the deoxyribofuranose ring. In addition, studies examining rates of sugar-aldehyde reduction and the sodium borohydride concentration dependence of the rate of formation of the covalent imine intermediate suggest that the reduction of the intermediate is rate-limiting in the reaction.     

Functional compromises among pH tolerance, site specificity, and sequence tolerance for a DNA-hydrolyzing deoxyribozyme

We recently reported the identification by in vitro selection of 10MD5, a deoxyribozyme that requires both Mn2+ and Zn2+ to hydrolyze a single-stranded DNA substrate with formation of 5′-phosphate and 3′-hydroxyl termini. DNA cleavage by 10MD5 proceeds with kobs=2.7 h(?1) and rate enhancement of 10(12) over the uncatalyzed P?O hydrolysis reaction. 10MD5 has a very sharp pH optimum near 7.5, with greatly reduced DNA cleavage rate and yield when the pH is changed by only 0.1 unit in either direction. Here we have optimized 10MD5 by reselection (in vitro evolution), leading to variants with broader pH tolerance, which is important for practical DNA cleavage applications. Because of the extensive Watson?Crick complementarity between deoxyribozyme and substrate, the parent 10MD5 is inherently sequence-specific; i.e., it is able to cleave one DNA substrate sequence in preference to other sequences. 10MD5 is also site-specific because only one phosphodiester bond within the DNA substrate is cleaved, although here we show that intentionally creating Watson?Crick mismatches near the cleavage site relaxes the site specificity. Newly evolved 10MD5 variants such as 9NL27 are also sequence-specific. However, the 9NL27 site specificity is relaxed for some substrate sequences even when full Watson?Crick complementarity is maintained, corresponding to a functional compromise between pH tolerance and site specificity. The site specificity of 9NL27 may be restored by expanding its “recognition site” from ATGT (as for 10MD5) to ATGTT or larger, i.e., by considering 9NL27 to have reduced substrate sequence tolerance relative to 10MD5. These findings provide fundamental insights into the interplay among key deoxyribozyme characteristics of tolerance and selectivity, with implications for ongoing development of practical DNA-catalyzed DNA hydrolysis.     

Molecular dynamics simulations investigation of neocarzinostatin chromophore-releasing pathways from the holo-NCS protein

The enediyne ring chromophore with strong DNA cleavage activity of neocarzinostatin is labile and therefore stabilization by forming the complex (carrying protein + chromophore: holo-NCS). Holo-NCS has gained much attention in clinical use as well as for drug delivery systems, but the chromophore-releasing mechanism to trigger binding to the target DNA with high affinity and producing DNA damage remain unclear. Three possible pathways were initially determined by conventional MD, essential dynamics and essential dynamics sampling. One of the paths runs along the naphthoate moiety; another runs along the amino sugar moiety; the third along the enediyne ring. Further, calculated forces and time by FPMD (force-probe molecular dynamics) suggest that the opening of the naphthoate moiety is most favorable pathway and Leu45, Phe76 and Phe78 all are key residues for chromophore release. In addition, conformational analyses indicate that the chromophore release is only local motions for the protein.     

Thermodynamics and kinetics of the cleavage of DNA catalyzed by bleomycin A5.

Microcalorimetry and UV-vis spectroscopy were used to conduct thermodynamic and kinetic investigations of the scission of calf thymus DNA catalyzed by bleomycin A5 (BLM-A5) in the presence of ferrous ion and oxygen. The molar reaction enthalpy for the cleavage, the Michaelis-Menten constant for calf thymus DNA and the turnover number of BLM-A5 were calculated by a novel thermokinetic method for an enzyme-catalyzed reaction to be -577 +/- 19 kJ.mol-1, 20.4 +/- 3.8 microm and 2.28 +/- 0.49 x 10-2 s-1, respectively, at 37.0 degrees C. This DNA cleavage was a largely exothermic reaction. The catalytic efficiency of BLM-A5 is of the same order of magnitude as that of lysozyme but several orders of magnitude lower than those of TaqI restriction endonuclease, NaeI endonuclease and BamHI endonuclease. By comparing the molar enthalpy change for the cleavage of calf thymus DNA induced by BLM-A5 with those for the scission of calf thymus DNA mediated by adriamycin and by (1,10-phenanthroline)-copper, it was found that BLM-A5 possessed the highest DNA cleavage efficiency among these DNA-damaging agents. These results suggest that BLM-A5 is not as efficient as a DNA-cleaving enzyme although the cleavage of DNA by BLM-A5 follows Michaelis-Menten kinetics. Binding of BLM-A5 to calf thymus DNA is driven by a favorable entropy increase with a less favorable enthalpy decrease, in line with a partial intercalation mode involved in BLM-catalyzed breakage of DNA.     

Intercalating polycyclic aromatic hydrocarbon-DNA adducts poison DNA religation by Vaccinia topoisomerase and act as roadblocks to digestion by exonuclease III

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts pervert the execution or fidelity of enzymatic DNA transactions and cause mutations and cancer. Here, we examine the effects of intercalating PAH-DNA adducts on the religation reaction of vaccinia DNA topoisomerase, a prototypal type IB topoisomerase (TopIB), and the 3' end-resection reaction of Escherichia coli exonuclease III (ExoIII), a DNA repair enzyme. Vaccinia TopIB forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p / N(-1) in duplex DNA. The rate of the forward cleavage reaction is suppressed to varying degrees by benzo[a]pyrene (BP) or benzo[c]phenanthrene (BPh) adducts at purine bases within the 3'-G(+5)G(+4)G(+3)A(+2)A(+1)T(-1)A(-2) sequence of the nonscissile strand. We report that BP adducts at the +1 and -2 N6-deoxyadenosine (dA) positions flanking the scissile phosphodiester slow the rate of DNA religation to a greater degree than they do the cleavage rate. By increasing the cleavage equilibrium constant > or = 10-fold, the BPdA adducts, which are intercalated via the major groove, act as TopIB poisons. With respect to ExoIII, we find that (i) single BPdA adducts act as durable roadblocks to ExoIII digestion, which is halted at sites 1 and 2 nucleotides prior to the modified base; (ii) single BPhdA adducts, which also intercalate via the major groove, elicit a transient pause prior to the lesion, which is eventually resected; and (iii) BPh adducts at N2-deoxyguanosine, which intercalate via the minor groove, are durable impediments to ExoIII digestion. These results highlight the sensitivity of repair outcomes to the structure of the PAH ring system and whether intercalation occurs via the major or minor groove.     

Microbial remediation of NTO in aqueous industrial wastes

The bioremediation of aqueous wastes containing 5-nitro-1,2,4-triazol-3-one (NTO) was investigated. The microorganism used is a Bacillus licheniformis strain, isolated from the contaminated solutions by enrichment techniques. The biodegradation was carried out in the waste (15 g l-1 NTO) and proceeded through the nitroreduction of NTO, followed by the ring cleavage of the formed primary amine 5-amino-1,2,4-triazol-3-one (ATO). Both steps were optimized and according to the optimal conditions, the nitroreduction of NTO is total in 24 h, while the degradation of ATO requires 2 weeks of incubation. The end products of the biodegradation were carbon dioxide (40%), urea and a polar compound, assumed to be hydroxyurea. A mechanism of ATO ring cleavage was postulated in the light of experimental data, and led us to propose an overall degradation sequence for NTO.     

Mechanistic studies of 1-aminocyclopropane-1-carboxylate deaminase: characterization of an unusual pyridoxal 5'-phosphate-dependent reaction

1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase (ACCD) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that cleaves the cyclopropane ring of ACC, to give α-ketobutyric acid and ammonia as products. The cleavage of the C(α)-C(β) bond of an amino acid substrate is a rare event in PLP-dependent enzyme catalysis. Potential chemical mechanisms involving nucleophile- or acid-catalyzed cyclopropane ring opening have been proposed for the unusual transformation catalyzed by ACCD, but the actual mode of cyclopropane ring cleavage remains obscure. In this report, we aim to elucidate the mechanistic features of ACCD catalysis by investigating the kinetic properties of ACCD from Pseudomonas sp. ACP and several of its mutant enzymes. Our studies suggest that the pK(a) of the conserved active site residue, Tyr294, is lowered by a hydrogen bonding interaction with a second conserved residue, Tyr268. This allows Tyr294 to deprotonate the incoming amino group of ACC to initiate the aldimine exchange reaction between ACC and the PLP coenzyme and also likely helps to activate Tyr294 for a role as a nucleophile to attack and cleave the cyclopropane ring of the substrate. In addition, solvent kinetic isotope effect (KIE), proton inventory, and (13)C KIE studies of the wild type enzyme suggest that the C(α)-C(β) bond cleavage step in the chemical mechanism is at least partially rate-limiting under k(cat)/K(m) conditions and is likely preceded in the mechanism by a partially rate-limiting step involving the conversion of a stable gem-diamine intermediate into a reactive external aldimine intermediate that is poised for cyclopropane ring cleavage. When viewed within the context of previous mechanistic and structural studies of ACCD enzymes, our studies are most consistent with a mode of cyclopropane ring cleavage involving nucleophilic catalysis by Tyr294.     

A new method for determining the stereochemistry of DNA cleavage reactions: application to the SfiI and HpaII restriction endonucleases and to the MuA transposase

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.