Position- and orientation-specific enhancement of topoisomerase I cleavage complexes by triplex DNA structures

Topoisomerase I (Top1) activities are sensitive to various endogenous base modifications, and anticancer drugs including the natural alkaloid camptothecin. Here, we show that triple helix-forming oligonucleotides (TFOs) can enhance Top1-mediated DNA cleavage by affecting either or both the nicking and the closing activities of Top1 depending on the position and the orientation of the triplex DNA structure relative to the Top1 site. TFO binding 1 bp downstream from the Top1 site enhances cleavage by inhibiting religation and to a lesser extent DNA nicking. In contrast, TFO binding 4 bp downstream from the Top1 site enhances DNA nicking especially when the 3′ end of the TFO is proximal to the Top1 site. However, when the orientation of the triplex is inverted, with its 5′ terminus 4 bp downstream from the Top1 site, religation is also inhibited. These position- and orientation-dependent effects of triplex structures on the Top1-mediated DNA cleavage and religation are discussed in the context of molecular modeling and effects of TFO on DNA twist and mobility at the duplex/triplex junction.     

Topoisomerase I Alone Is Sufficient to Produce Short DNA Deletions and Can Also Reverse Nicks at Ribonucleotide Sites

Ribonucleotide monophosphates (rNMPs) are among the most frequent form of DNA aberration, as high ratios of ribonucleotide triphosphate:deoxyribonucleotide triphosphate pools result in approximately two misincorporated rNMPs/kb of DNA. The main pathway for the removal of rNMPs is by RNase H2. However, in a RNase H2 knock-out yeast strain, a topoisomerase I (Top1)-dependent mutator effect develops with accumulation of short deletions within tandem repeats. Proposed models for these deletions implicated processing of Top1-generated nicks at rNMP sites and/or sequential Top1 binding, but experimental support has been lacking thus far. Here, we investigated the biochemical mechanism of the Top1-induced short deletions at the rNMP sites by generating nicked DNA substrates bearing 2′,3′-cyclic phosphates at the nick sites, mimicking the Top1-induced nicks. We demonstrate that a second Top1 cleavage complex adjacent to the nick and subsequent faulty Top1 religation led to the short deletions. Moreover, when acting on the nicked DNA substrates containing 2′,3′-cyclic phosphates, Top1 generated not only the short deletion, but also a full-length religated DNA product. A catalytically inactive Top1 mutant (Top1-Y723F) also induced the full-length products, indicating that Top1 binding independent of its enzymatic activity promotes the sealing of DNA backbones via nucleophilic attacks by the 5′-hydroxyl on the 2′,3′-cyclic phosphate. The resealed DNA would allow renewed attempt for repair by the error-free RNase H2-dependent pathway in vivo. Our results provide direct evidence for the generation of short deletions by sequential Top1 cleavage events and for the promotion of nick religation at rNMP sites by Top1.     

A small organic compound enhances the religation reaction of human topoisomerase I and identifies crucial elements for the religation mechanism

The different steps of the human Top1 (topoisomerase I) catalytic cycle have been analysed in the presence of a pentacyclic-diquinoid synthetic compound. The experiments indicate that it efficiently inhibits the cleavage step of the enzyme reaction, fitting well into the catalytic site. Surprisingly the compound, when incubated with the binary topoisomerase–DNA cleaved complex, helps the enzyme to remove itself from the cleaved DNA and close the DNA gap, increasing the religation rate. The compound also induces the religation of the stalled enzyme–CPT (camptothecin)–DNA ternary complex. Analysis of the molecule docked over the binary complex, together with its chemical properties, suggests that the religation enhancement is due to the presence on the compound of two oxygen atoms that act as hydrogen acceptors. This property facilitates the deprotonation of the 5′ DNA end, suggesting that this is the limiting step in the topoisomerase religation mechanism.     

Cross talk between the +73/294 interaction and the cleavage site in RNase P RNA mediated cleavage

To monitor functionally important metal ions and possible cross talk in RNase P RNA mediated cleavage we studied cleavage of substrates, where the 2′OH at the RNase P cleavage site (at −1) and/or at position +73 had been replaced with a 2′ amino group (or 2′H). Our data showed that the presence of 2′ modifications at these positions affected cleavage site recognition, ground state binding of substrate and/or rate of cleavage. Cleavage of 2′ amino substituted substrates at different pH showed that substitution of Mg²⁺ by Mn²⁺ (or Ca²⁺), identity of residues at and near the cleavage site, and addition of C5 protein influenced the frequency of miscleavage at −1 (cleavage at the correct site is referred to as +1). From this we infer that these findings point at effects mediated by protonation/deprotonation of the 2′ amino group, i.e. an altered charge distribution, at the site of cleavage. Moreover, our data suggested that the structural architecture of the interaction between the 3′ end of the substrate and RNase P RNA influence the charge distribution at the cleavage site as well as the rate of cleavage under conditions where the chemistry is suggested to be rate limiting. Thus, these data provide evidence for cross talk between the +73/294 interaction and the cleavage site in RNase P RNA mediated cleavage. We discuss the role metal ions might play in this cross talk and the likelihood that at least one functionally important metal ion is positioned in the vicinity of, and use the 2′OH at the cleavage site as an inner or outer sphere ligand.     

On the structural basis and design guidelines for type II topoisomerase-targeting anticancer drugs

Type II topoisomerases (Top2s) alter DNA topology via the formation of an enzyme–DNA adduct termed cleavage complex, which harbors a transient double-strand break in one DNA to allow the passage of another. Agents targeting human Top2s are clinically active anticancer drugs whose trapping of Top2-mediated DNA breakage effectively induces genome fragmentation and cell death. To understand the structural basis of this drug action, we previously determined the structure of human Top2 β-isoform forming a cleavage complex with the drug etoposide and DNA, and described the insertion of drug into DNA cleavage site and drug-induced decoupling of catalytic groups. By developing a post-crystallization drug replacement procedure that simplifies structural characterization of drug-stabilized cleavage complexes, we have extended the analysis toward other structurally distinct drugs, m-AMSA and mitoxantrone. Besides the expected drug intercalation, a switch in ribose puckering in the 3′-nucleotide of the cleavage site was robustly observed in the new structures, representing a new mechanism for trapping the Top2 cleavage complex. Analysis of drug-binding modes and the conformational landscapes of the drug-binding pockets provide rationalization of the drugs’ structural-activity relationships and explain why Top2 mutants exhibit differential effects toward each drug. Drug design guidelines were proposed to facilitate the development of isoform-specific Top2-targeting anticancer agents.     

Cleavage of deoxyoxanosine-containing oligodeoxyribonucleotides by bacterial endonuclease V

Oxanine (O) is a deamination product derived from guanine with the nitrogen at the N¹ position substituted by oxygen. Cytosine, thymine, adenine, guanine as well as oxanine itself can be incorporated by Klenow Fragment to pair with oxanine in a DNA template with similar efficiency, indicating that oxanine in DNA may cause various mutations. As a nucleotide, deoxyoxanosine may substitute for deoxyguanosine to complete a primer extension reaction. Endonuclease V, an enzyme known for its enzymatic activity on uridine-, inosine- and xanthosine-containing DNA, can cleave oxanosine-containing DNA at the second phosphodiester bond 3′ to the lesion. Mg²⁺ or Mn²⁺, and to a small extent Co²⁺ or Ni²⁺, support the oxanosine-containing DNA cleavage activity. All four oxanosine-containing base pairs (A/O, T/O, C/O and G/O) were cleaved with similar efficiency. The cleavage of double-stranded oxanosine-containing DNA was ~6-fold less efficient than that of double-stranded inosine-containing DNA. Single-stranded oxanosine-containing DNA was cleaved with a lower efficiency as compared with double-stranded oxanosine-containing DNA. A metal ion enhances the binding of endonuclease V to double-stranded and single-stranded oxanosine-containing DNA 6- and 4-fold, respectively. Hypothetic models of oxanine-containing base pairs and deaminated base recognition mechanism are presented.     

Substitution of conserved residues within the active site alters the cleavage religation equilibrium of DNA topoisomerase I

Eukaryotic DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of camptothecin (CPT). Mutation of conserved residues in close proximity to the active site tyrosine (Tyr(727) of yeast Top1p) alters the DNA cleavage religation equilibrium, inducing drug-independent cell lethality. Previous studies indicates that yeast Top1T722Ap and Top1N726Hp cytotoxicity results from elevated levels of covalent enzyme-DNA intermediates. Here we show that Top1T722Ap acts as a CPT mimetic by exhibiting reduced rates of DNA religation, whereas increased Top1N726Hp.DNA complexes result from elevated DNA binding and cleavage. We also report that the combination of the T722A and N726H mutations in a single protein potentiates the cytotoxic action of the enzyme beyond that induced by co-expression of the single mutants. Moreover, the addition of CPT to cells expressing the double top1T722A/N726H mutant did not enhance cell lethality. Thus, independent alterations in DNA cleavage and religation contribute to the lethal phenotype. The formation of distinct cytotoxic lesions was also evidenced by the different responses induced by low levels of these self-poisoning enzymes in isogenic strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis. Substitution of Asn(726) with Phe or Tyr also produces self-poisoning enzymes, implicating stacking interactions in the increased kinetics of DNA cleavage by Top1N726Hp and Top1N726Fp. In contrast, replacing the amide side chain of Asn(726) with Gln renders Top1N726Qp resistant to CPT, suggesting that the orientation of the amide within the active site is critical for effective CPT binding.     

Active site constraints in the hydrolysis reaction catalyzed by bacterial RNase P: analysis of precursor tRNAs with a single 3'-S-phosphorothiolate internucleotide linkage

Endonucleolytic processing of precursor tRNAs (ptRNAs) by RNase P yields 3′-OH and 5′-phosphate termini, and at least two metal ions are thought to be essential for catalysis. To determine if the hydrolysis reaction catalyzed by bacterial RNase P (RNAs) involves stabilization of the 3′-oxyanion leaving group by direct coordination to one of the catalytic metal ions, ptRNA substrates with single 3′-S-phosphorothiolate linkages at the RNase P cleavage site were synthesized. With a 3′-S-phosphorothiolate-modified ptRNA carrying a 7 nt 5′-flank, a complete shift of the cleavage site to the next unmodified phosphodiester in the 5′-direction was observed. Cleavage at the modified linkage was not restored in the presence of thiophilic metal ions, such as Mn²⁺ or Cd²⁺. To suppress aberrant cleavage, we also constructed a 3′-S-phosphorothiolate-modified ptRNA with a 1 nt 5′-flank. No detectable cleavage of this substrate was seen in reactions catalyzed by RNase P RNAs from Escherichia coli and Bacillus subtilis, independent of the presence of thiophilic metal ions. Ground state binding of modified ptRNAs was not impaired, suggesting that the 3′-S-phosphorothiolate modification specifically prevents formation of the transition state, possibly by excluding catalytic metal ions from the active site.     

Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site

The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo⁻) and DNA polymerase β (pol β) using ¹⁹F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol β. The addition of a non-hydrolysable 2′-deoxycytosine-5′-[(α,β)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo⁻ complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol β, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with ¹⁹F NMR data. Surface plasmon resonance binding kinetics revealed that pol β binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential.     

Effect of manganese on in vitro replication of damaged DNA catalyzed by the herpes simplex virus type-1 DNA polymerase

In vitro bypass of damaged DNA by replicative DNA polymerases is usually blocked by helix-distorting or bulky DNA lesions. In this study, we report that substitution of the divalent metal ion Mg²⁺ with Mn²⁺ promotes quantitative replication of model DNA substrates containing the major cisplatin or N-2-acetylaminofluorene adducts by the catalytic subunit (UL30) of the replicative DNA polymerase of herpes simplex virus. The ability of Mn²⁺ ions to confer bypass of bulky lesions was not observed with other replicative DNA polymerases of the B family, such as bacteriophage T4 or δ polymerases. However, for these enzymes, manganese induced the incorporation of one nucleotide opposite the first (3′) guanine of the d(GpG) intrastrand cisplatin lesion. Translesion replication of the cisplatin adduct by UL30 led to the incorporation of mismatched bases, with the preferential incorporation of dAMP opposite the 3′ guanine of the lesion. Furthermore, substitution of MgCl₂ with MnCl₂ greatly inhibited the 3′ to 5′ exonuclease of UL30 but had a far lesser effect on that of T4 DNA polymerase. Finally, manganese induced a conformational change in the structure of UL30 bound to the platinated substrate. Taken together, the latter findings suggest a mechanism by which manganese might allow UL30 to efficiently promote translesion DNA synthesis in vitro.     

Unusually wide co-factor tolerance in a metalloenzyme; divalent metal ions modulate endo–exonuclease activity in T5 exonuclease

T5 5′–3′ exonuclease is a member of a homologous group of 5′ nucleases which require divalent metal co-factors. Structural and biochemical studies suggest that single-stranded DNA substrates thread through a helical arch or hole in the protein, thus bringing the phosphodiester backbone into close proximity with the active site metal co-factors. In addition to the expected use of Mg²⁺, Mn²⁺ and Co²⁺ as co-factors, we found that divalent zinc, iron, nickel and copper ions also supported catalysis. Such a range of co-factor utilisation is unusual in a single enzyme. Some co-factors such as Mn²⁺ stimulated the cleavage of double-stranded closed-circular plasmid DNA. Such endonucleolytic cleavage of circular double-stranded DNA cannot be readily explained by the threading model proposed for the cleavage of substrates with free 5′-ends as the hole observed in the crystal structure of T5 exonuclease is too small to permit the passage of double-stranded DNA. We suggest that such a substrate may gain access to the active site of the enzyme by a process which does not involve threading.     

Alterations in linker flexibility suppress DNA topoisomerase I mutant-induced cell lethality

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of a covalent enzyme-DNA intermediate, which is reversibly stabilized by the anticancer agent camptothecin (CPT). Crystallographic studies of the 70-kDa C terminus of human Top1p bound to duplex DNA describe a monomeric protein clamp circumscribing the DNA helix. The structures, which lack the N-terminal domain, comprise the conserved clamp, an extended linker domain, and the conserved C-terminal active site Tyr domain. CPT bound to the covalent Top1p-DNA complex limits linker flexibility, allowing structural determination of this domain. We previously reported that mutation of Ala(653) to Pro in the linker increases the rate of enzyme-catalyzed DNA religation, thereby rendering Top1A653Pp resistant to CPT (Fiorani, P., Bruselles, A., Falconi, M., Chillemi, G., Desideri, A., and Benedetti P. (2003) J. Biol. Chem. 278, 43268-43275). Molecular dynamics studies suggested mutation-induced increases in linker flexibility alter Top1p catalyzed DNA religation. To address the functional consequences of linker flexibility on enzyme catalysis and drug sensitivity, we investigated the interactions of the A653P linker mutation with a self-poisoning T718A mutation within the active site of Top1p. The A653P mutation suppressed the lethal phenotype of Top1T718Ap in yeast, yet did not restore enzyme sensitivity to CPT. However, the specific activity of the double mutant was decreased in vivo and in vitro, consistent with a decrease in DNA binding. These findings support a model where changes in the flexibility or orientation of the linker alter the geometry of the active site and thereby the kinetics of DNA cleavage/religation catalyzed by Top1p.     

Sequence-function relationships provide new insight into the cleavage site selectivity of the 8-17 RNA-cleaving deoxyribozyme

Many sequence variations of the 8–17 RNA-cleaving deoxyribozyme have been isolated through in vitro selection. In an effort to understand how these sequence variations affect cleavage site selectivity, we systematically mutated the catalytic core of 8–17 and measured the cleavage activity of each mutant deoxyribozyme against all 16 possible chimeric (RNA/DNA) dinucleotide junctions. We observed sequence-function relationships that suggest how the following non-conserved positions in the catalytic core influence selectivity at the dinucleotide (5′ rN₁₈-N_1.1 3′) cleavage site: (i) positions 2.1 and 12 represent a primary determinant of the selectivity at the 3′ position (N_1.1) of the cleavage site; (ii) positions 15 and 15.0 represent a primary determinant of the selectivity at the 5′ position (rN₁₈) of the cleavage site and (iii) the sequence of the 3-bp intramolecular stem has relatively little influence on cleavage site selectivity. Furthermore, we report for the first time that 8–17 variants have the collective ability to cleave all dinucleotide junctions with rate enhancements of at least 1000-fold over background. Three optimal 8–17 variants, identified from ~75 different sequences that were examined, can collectively cleave 10 of 16 junctions with useful rates of ≥0.1 min⁻¹, and exhibit an overall hierarchy of reactivity towards groups of related junctions according to the order NG > NA > NC > NT.     

Mutation of Gly721 alters DNA topoisomerase I active site architecture and sensitivity to camptothecin

DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA via a concerted mechanism of DNA strand cleavage and religation. Top1p is the cellular target of the anti-cancer drug camptothecin (CPT), which reversibly stabilizes a covalent enzyme-DNA intermediate. Top1p clamps around duplex DNA, wherein the core and C-terminal domains are connected by extended alpha-helices (linker domain), which position the active site Tyr of the C-terminal domain within the catalytic pocket. The physical connection of the linker with the Top1p clamp as well as linker flexibility affect enzyme sensitivity to CPT. Crystallographic data reveal that a conserved Gly residue (located at the juncture between the linker and C-terminal domains) is at one end of a short alpha-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this alpha-helix extends to include Gly and the preceding Leu. We report that mutation of this conserved Gly in yeast Top1p alters enzyme sensitivity to CPT. Mutating Gly to Asp, Glu, Asn, Gln, Leu, or Ala enhanced enzyme CPT sensitivity, with the acidic residues inducing the greatest increase in drug sensitivity in vivo and in vitro. By contrast, Val or Phe substituents rendered the enzyme CPT-resistant. Mutation-induced alterations in enzyme architecture preceding the active site Tyr suggest these structural transitions modulate enzyme sensitivity to CPT, while enhancing the rate of DNA cleavage. We postulate that this conserved Gly residue provides a flexible hinge within the Top1p catalytic pocket to facilitate linker dynamics and the structural alterations that accompany drug binding of the covalent enzyme-DNA intermediate.     

Mechanistic aspects of CoII(HAPP)(TFA)2 in DNA bulge-specific recognition

A novel octahedral complex Co^II(HAPP)(TFA)₂ [hexaazaphenantholine-cyclophane (HAPP), trifluoroacetate (TFA)] is a DNA bulge-specific probe with single-strand DNA cleavage activity in the presence of H₂O₂. This complex exhibits low affinity towards double-stranded DNA and low reactivity toward single-stranded DNA. Metal–HAPP complexes with different coordination number and ring size were synthesized and their selectivity and reactivity for DNA bulges were compared. The DNA sequence at the bulge site influences the intensity of cleavage at the bulge and the flanking sites after piperidine treatment. Cleavage specificity of Co^II(HAPP)(TFA)₂ was characterized extensively using scavenger reagents to quench the cleavage reaction and high-resolution polyacrylamide gel electrophoresis. In addition, 3′-phosphoglycolate cleavage products were trapped and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. These data were used to deduce that the DNA cleavage pathway for Co^IIHAPP²⁺ in the presence of H₂O₂ involves 4′-H abstraction of the deoxyribose moiety.     

Base-displaced intercalation of the 2-amino-3-methylimidazo[4,5-f]quinolone N2-dG adduct in the NarI DNA recognition sequence

2-Amino-3-methylimidazo[4,5-f]quinolone (IQ), a heterocyclic amine found in cooked meats, undergoes bioactivation to a nitrenium ion, which alkylates guanines at both the C8-dG and N²-dG positions. The conformation of a site-specific N²-dG-IQ adduct in an oligodeoxynucleotide duplex containing the iterated CG repeat restriction site of the NarI endonuclease has been determined. The IQ moiety intercalates, with the IQ H4a and CH₃ protons facing the minor groove, and the IQ H7a, H8a and H9a protons facing the major groove. The adducted dG maintains the anti-conformation about the glycosyl bond. The complementary dC is extruded into the major groove. The duplex maintains its thermal stability, which is attributed to stacking between the IQ moiety and the 5′- and 3′-neighboring base pairs. This conformation is compared to that of the C8-dG-IQ adduct in the same sequence, which also formed a ‘base-displaced intercalated’ conformation. However, the C8-dG-IQ adopted the syn conformation placing the Watson−Crick edge of the modified dG into the major groove. In addition, the C8-dG-IQ adduct was oriented with the IQ CH₃ group and H4a and H5a facing the major groove. These differences may lead to differential processing during DNA repair and replication.     

Features of Pro-σK Important for Cleavage by SpoIVFB,an Intramembrane Metalloprotease

Intramembrane proteases regulate diverse processes by cleaving substrates within a transmembrane segment or near the membrane surface. Bacillus subtilis SpoIVFB is an intramembrane metalloprotease that cleaves Pro-σ^K during sporulation. To elucidate features of Pro-σ^K important for cleavage by SpoIVFB, coexpression of the two proteins in Escherichia coli was used along with cell fractionation. In the absence of SpoIVFB, a portion of the Pro-σ^K was peripherally membrane associated. This portion was not observed in the presence of SpoIVFB, suggesting that it serves as the substrate. Deletion of Pro-σ^K residues 2 to 8, addition of residues at its N terminus, or certain single-residue substitutions near the cleavage site impaired cleavage. Certain multiresidue substitutions near the cleavage site changed the position of cleavage, revealing preferences for a small residue preceding the cleavage site N-terminally (i.e., at the P1 position) and a hydrophobic residue at the second position following the cleavage site C-terminally (i.e., P2′). These features appear to be conserved among Pro-σ^K orthologs. SpoIVFB did not tolerate an aromatic residue at P1 or P2′ of Pro-σ^K. A Lys residue at P3′ of Pro-σ^K could not be replaced with Ala unless a Lys was provided farther C-terminally (e.g., at P9′). α-Helix-destabilizing residues near the cleavage site were not crucial for SpoIVFB to cleave Pro-σ^K. The preferences and tolerances of SpoIVFB are somewhat different from those of other intramembrane metalloproteases, perhaps reflecting differences in the interaction of the substrate with the membrane and the enzyme.     

Quantitative studies of Mn(2+)-promoted specific and non-specific cleavages of a large RNA: Mn(2+)-GAAA ribozymes and the evolution of small ribozymes

Manganese (Mn²⁺) promotes specific cleavage at two major (I and III) and four minor (II, IV, V and VI) sites, in addition to slow non-specific cleavage, in a 659-nucleotide RNA containing the Cr.LSU group I intron. The specific cleavages occurred between G and AAA sequences and thus can be considered Mn²⁺-GAAA ribozymes. We have estimated rates of specific and non-specific cleavages under different conditions. Comparisons of the rates of major-specific and background cleavages gave a maximal specificity of approximately 900 for GAAA cleavage. Both specific and non-specific cleavages showed hyperbolic kinetics and there was no evidence of cooperativity with Mn²⁺ concentration. Interestingly, at site III, Mg²⁺ alone promoted weak, but the same specific cleavage as Mn²⁺. When added with Mn²⁺, Mg²⁺ had a synergistic effect on cleavage at site III, but inhibited cleavage at the other sites. Mn²⁺ cleavage at site III also exhibited lower values of K (Mn²⁺ requirement), pH-dependency and activation energy than did cleavage at the other sites. In contrast, the pH-dependency and activation energy for cleavage at site I was similar to non-specific cleavage. These results increase our understanding of the Mn²⁺-GAAA ribozyme. The implications for evolution of small ribozymes are also discussed.     

Two novel dATP analogs for DNA photoaffinity labeling

Two new photoreactive dATP analogs, N⁶-[4-azidobenzoyl–(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (AB-dATP) and N⁶-[4-[3-(trifluoromethyl)-diazirin-3-yl]benzoyl-(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (DB-dATP), were synthesized from 2′-deoxyadenosine-5′-monophosphate in a six step procedure. Synthesis starts with aminoethylation of dAMP and continues with rearrangement of N¹-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate to N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate (N⁶-dAMP). Next, N⁶-dAMP is converted into the triphosphate form by first protecting the N-6 primary amino group before coupling the pyrophosphate. After pyrophosphorylation, the material is deprotected to yield N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-triphosphate (N⁶-dATP). The N-6 amino group is subsequently used to attach either a phenylazide or phenyldiazirine and the photoreactive nucleotide is then enzymatically incorporated into DNA. N⁶-dATP and its photoreactive analogs AB-dATP and DB-dATP were successfully incorporated into DNA using the exonuclease-free Klenow fragment of DNA polymerase I in a primer extension reaction. UV irradiation of the primer extension reaction with AB-dATP or DB-dATP showed specific photocrosslinking of DNA polymerase I to DNA.     

Cation-dependent cleavage of the duplex form of the subtype-B HIV-1 RNA dimerization initiation site

The crystal structure of subtype-B HIV-1 genomic RNA Dimerization Initiation Site duplex revealed chain cleavage at a specific position resulting in 3′-phosphate and 5′-hydroxyl termini. A crystallographic analysis showed that Ba²⁺, Mn²⁺, Co²⁺ and Zn²⁺ bind specifically on a guanine base close to the cleaved position. The crystal structures also point to a necessary conformational change to induce an ‘in-line’ geometry at the cleavage site. In solution, divalent cations increased the rate of cleavage with pH/pKa compensation, indicating that a cation-bound hydroxide anion is responsible for the cleavage. We propose a ‘Trojan horse’ mechanism, possibly of general interest, wherein a doubly charged cation hosted near the cleavage site as a ‘harmless’ species is further transformed in situ into an ‘aggressive’ species carrying a hydroxide anion.