Protection of all cleavable sites of DNA with the multiple CGCG or continuous CGG sites from the restriction enzyme, indicative of simultaneous binding of small ligands.

The anthracenone ligands (1-12) with a keto-phenol and a hydroxamic acid unit were synthesized and evaluated by a restriction enzyme inhibition assay. DNA substrates composed of multiple CGCG or CGG sites are fully hydrolyzed by a restriction enzyme that is selective for each sequence. Under such conditions, the full-length DNA substrate remains only when the ligand binds to all binding sites and protects it from hydrolysis by the restriction enzymes. In the assay using AccII and the 50-mer DNA substrates containing a different number of CGCG sites at different non-binding AT base pair intervals, the more the CGCG sites, the more the full-length DNA increased. Namely, simultaneous binding of the ligand (5) to the CGCG sites increased in the order of (CGCG)5>(CGCG)2>(CGCG)1. Furthermore, the length of the spacer of the hydroxamic acid to the anthracenone skeleton played an important role in the preference for the number of the d(A/T) base pairs between the CGCG sites. The long spacer-ligand (5) showed a preference to the CGCG sites with five AT pairs, and the short spacer-ligand (10) to that with two AT pairs. The ligand (12) with the shortest spacer showed a preference in simultaneous binding to the 54-mer DNA composed of 16 continuous CGG sites in the assay using the restriction enzyme Fnu4HI that hydrolyzes the d(GCGGC)/d(CGCCG) site. Application of these ligands to biological systems including the repeat DNA sequence should be of significant interest.

RNA aptamer inhibitors of a restriction endonuclease.

Restriction endonucleases (REases) recognize and cleave short palindromic DNA sequences, protecting bacterial cells against bacteriophage infection by attacking foreign DNA. We are interested in the potential of folded RNA to mimic DNA, a concept that might be applied to inhibition of DNA-binding proteins. As a model system, we sought RNA aptamers against the REases BamHI, PacI and KpnI using systematic evolution of ligands by exponential enrichment (SELEX). After 20 rounds of selection under different stringent conditions, we identified the 10 most enriched RNA aptamers for each REase. Aptamers were screened for binding and specificity, and assayed for REase inhibition. We obtained eight high-affinity (Kd ∼12-30 nM) selective competitive inhibitors (IC50 ∼20-150 nM) for KpnI. Predicted RNA secondary structures were confirmed by in-line attack assay and a 38-nt derivative of the best anti-KpnI aptamer was sufficient for inhibition. These competitive inhibitors presumably act as KpnI binding site analogs, but lack the primary consensus KpnI cleavage sequence and are not cleaved by KpnI, making their potential mode of DNA mimicry fascinating. Anti-REase RNA aptamers could have value in studies of REase mechanism and may give clues to a code for designing RNAs that competitively inhibit DNA binding proteins including transcription factors.

Iron(II) supramolecular helicates condense plasmid DNA and inhibit vital DNA-related enzymatic activities.

The dinuclear iron(II) supramolecular helicates [Fe2 L3 ]Cl4 (L=C25 H20 N4 ) bind to DNA through noncovalent (i.e., hydrogen-bonding, electrostatic) interactions and exhibit antimicrobial and anticancer effects. In this study, we show that the helicates condense plasmid DNA with a much higher potency than conventional DNA-condensing agents. Notably, molecules of DNA in the presence of the M enantiomer of [Fe2 L3 ]Cl4 do not form intermolecular aggregates typically formed by other condensing agents, such as spermidine or spermine. The helicates inhibit the activity of several DNA-processing enzymes, such as RNA polymerase, DNA topoisomerase I, deoxyribonuclease I, and site-specific restriction endonucleases. However, the results also indicate that the DNA condensation induced by the helicates does not play a crucial role in these inhibition reactions. The mechanisms for the inhibitory effects of [Fe2 L3 ]Cl4 helicates on DNA-related enzymatic activities have been proposed.

[Antirestriction activity of T7 Ocr protein in monomeric and dimeric forms].

The Ocr protein, encoded by 0.3 (ocr) gene of bacteriophage T7, belongs to the family of antirestriction proteins that specifically inhibit the type I restriction-modification systems. Native Ocr forms homodimer (Ocr)2 both in solution and in the crystalline state. The Ocr protein belongs to the family of mimicry proteins. F53D A57E and E53R V77D mutant proteins were obtained, which form monomers. It was shown that the values of the dissociation constants Kd for Ocr, Ocr F53D A57E and Ocr F53RV77D proteins with EcoKI enzyme differ in 1000 times: Kd (Ocr) = 10(-10) M, Kd (Ocr F53D A57E and Ocr F53R V77D) = 10(-7) M. Antimodification activity of the Ocr monomeric forms is significantly reduced. We have shown, that Ocr dimeric form has fundamental importance for high inhibitory activity.

Dependence of DNA-protein cross-linking via guanine oxidation upon local DNA sequence as studied by restriction endonuclease inhibition.

Oxidative damage plays a causative role in many diseases, and DNA-protein cross-linking is one important consequence of such damage. It is known that GG and GGG sites are particularly prone to one-electron oxidation, and here we examined how the local DNA sequence influences the formation of DNA-protein cross-links induced by guanine oxidation. Oxidative DNA-protein cross-linking was induced between DNA and histone protein via the flash quench technique, a photochemical method that selectively oxidizes the guanine base in double-stranded DNA. An assay based on restriction enzyme cleavage was developed to detect the cross-linking in plasmid DNA. Following oxidation of pBR322 DNA by flash quench, several restriction enzymes (PpuMI, BamHI, EcoRI) were then used to probe the plasmid surface for the expected damage at guanine sites. These three endonucleases were strongly inhibited by DNA-protein cross-linking, whereas the AT-recognizing enzyme AseI was unaffected in its cleavage. These experiments also reveal the susceptibility of different guanine sites toward oxidative cross-linking. The percent inhibition observed for the endonucleases, and their pBR322 cleavage sites, decreased in the order: PpuMI (5'-GGGTCCT-3' and 5'-AGGACCC-3') > BamHI (5'-GGATCC-3') > EcoRI (5'-GAATTC-3'), a trend consistent with the observed and predicted tendencies for guanine to undergo one-electron oxidation: 5'-GGG-3' > 5'-GG-3' > 5'-GA-3'. Thus, it appears that in mixed DNA sequences the guanine sites most vulnerable to oxidative cross-linking are those that are easiest to oxidize. These results further indicate that equilibration of the electron hole in the plasmid DNA occurs on a time scale faster than that of cross-linking.

A mutational analysis of DNA mimicry by ocr, the gene 0.3 antirestriction protein of bacteriophage T7.

The ocr protein of bacteriophage T7 is a structural and electrostatic mimic of approximately 24 base pairs of double-stranded B-form DNA. As such, it inhibits all Type I restriction and modification (R/M) enzymes by blocking their DNA binding grooves and inactivates them. This allows the infection of the bacterial cell by T7 to proceed unhindered by the action of the R/M defence system. We have mutated aspartate and glutamate residues on the surface of ocr to investigate their contribution to the tight binding between the EcoKI Type I R/M enzyme and ocr. Contrary to expectations, all of the single and double site mutations of ocr constructed were active as anti-R/M proteins in vivo and in vitro indicating that the mimicry of DNA by ocr is very resistant to change.

[Definition of the specificity of DNA-methyltransferase M.Bsc4I in cell lysate by blocking of restriction endonucleases and computer modeling].

The specificity of DNA-methyltransferase M.Bsc4I was defined in cellular lysate of Bacillus schlegelii 4. For this purpose, we used methylation sensitivity of restriction endonucleases, and also modeling of methylation. The modeling consisted in editing sequences of DNA using replacements of methylated bases and their complementary bases. The substratum DNA processed by M.Bsc4I also were used for studying sensitivity of some restriction endonucleases to methylation. Thus, it was shown that M.Bsc4I methylated 5'-Cm4CNNNNNNNGG-3' and the overlapped dcm-methylation blocked its activity. The offered approach can appear universal enough and simple for definition of specificity of DNA-methyltransferases.

Antirestriction activities of KlcA (RP4) and ArdB (R64) proteins.

Antirestriction proteins of the ArdB group (ArdB, KlcA) specifically inhibit restriction (endonuclease) activity of restriction-modification (RM) type I systems. Antirestriction activity of KlcA and ArdB, encoded in transmissible plasmids RP4 (IncPα) and R64 (IncI1), respectively, has been determined. We show that the protein KlcA (RP4), an amino acid sequence identical to that of the protein KlcA (RK2), inhibits the activity of EcoKI when the klcA gene is located on the plasmid under the control of strong promoter. It was demonstrated that proteins KlcA (RP4) and ArdB (R64) are characterized by approximately equal antirestriction activity. Analysis of amino acid sequences of ArdB homologs revealed four groups of conserved amino acids located on the surface of the protein globule: (1) R16, E32, W51; (2) Y46, G48; (3) S84, D86, E132 and (4) N77, L140, D141. It was shown that substitution of polar amino acids to hydrophobic A and L leads to a significant decrease in the ArdB antirestriction activity level (approximately 100-fold). A conserved region forming a 'ring belt' on the globule surface consisting of E32, S84, E132, and both N77 and D141 as the 'key section' of ArdB/KlcA was identified.

New peptide inhibitors of type IB topoisomerases: similarities and differences vis-a-vis inhibitors of tyrosine recombinases.

Topoisomerases relieve topological tension in DNA by breaking and rejoining DNA phosphodiester bonds. Type IB topoisomerases such as vaccinia topoisomerase (vTopo) and human topoisomerase I are structurally and mechanistically similar to the tyrosine recombinase family of enzymes, which includes bacteriophage lambda Integrase (Int). Previously, our laboratory identified peptide inhibitors of Int from a synthetic peptide combinatorial library. The most potent of these peptides also inhibit vTopo. Here, we used the same mixture-based screening procedure to identify peptide inhibitors directly against vTopo using a plasmid relaxation assay. The two most potent new peptides identified, WYCRCK and KCCRCK, inhibit plasmid relaxation, DNA cleavage and Holliday junction (HJ) resolution mediated by vTopo. The peptides tested bind double-stranded DNA at high concentrations but do not appear to displace the enzyme from its DNA substrate. WYCRCK binds specifically to HJ and perturbs the central base-pairing. This peptide also accumulates HJ intermediates when it inhibits Int-mediated recombination, whereas KCCRCK does not. Interestingly, WYCRCK shares four amino acids with a peptide identified against Int, WRWYCR. The octapeptide WRWYCRCK, containing amino acids from both hexapeptides, is more potent than either against vTopo. All peptides are less potent against the type IA Escherichia coli topoisomerase I or against restriction endonucleases. Like the Int-inhibitory peptide WRWYCR, WYCRCK binds to HJs, and both inhibit junction resolution by vTopo. Our results suggest that the newly identified WYCRCK and peptide WRWYCR interact with a distorted DNA intermediate arising during vTopo-mediated catalysis, or interfere with specific interactions between vTopo and DNA.

Determination of restriction enzyme activity when cutting DNA labeled with the TOTO dye family.

Optical mapping, a single DNA molecule genome analysis platform that can determine methylation profiles, uses fluorescently labeled DNA molecules that are elongated on the surface and digested with a restriction enzyme to produce a barcode of that molecule. Understanding how the cyanine fluorochromes affect enzyme activity can lead to other fluorochromes used in the optical mapping system. The effects of restriction digestion on fluorochrome labeled DNA (Ethidium Bromide, DAPI, H33258, EthD-1, TOTO-1) have been analyzed previously. However, TOTO-1 is a part of a family of cyanine fluorochromes (YOYO-1, TOTO-1, BOBO-1, POPO-1, YOYO-3, TOTO-3, BOBO-3, and POPO-3) and the rest of the fluorochromes have not been examined in terms of their effects on restriction digestion. In order to determine if the other dyes in the TOTO-1 family inhibit restriction enzymes in the same way as TOTO-1, lambda DNA was stained with a dye from the TOTO family and digested. The restriction enzyme activity in regards to each dye, as well as each restriction enzyme, was compared to determine the extent of digestion. YOYO-1, TOTO-1, and POPO-1 fluorochromes inhibited ScaI-HF, PmlI, and EcoRI restriction enzymes. Additionally, the mobility of labeled DNA fragments in an agarose gel changed depending on which dye was intercalated.

[Antirestriction and antimodification activities of the ArdA protein encoded by the IncI1 transmissive plasmids R-64 and ColIb-P9].

Proteins of the Ard family are specific inhibitors of type I restriction-modification enzymes. The ArdA of R64 is highly homologous to ColIb-P9 ArdA, differing only by four amino acid residues of the overall 166. However, unlike ColIb-P9 ArdA, which inhibits both the endonuclease and the methylase activities of EcoKI, the R64 ArdA protein inhibits only the endonuclease activity of this enzyme. The mutant forms of R64 ArdA--A29T, S43A, and Y75W, capable of partially reversing the protein to ColIb-P9 ArdA form--were produced by directed mutagenesis. It was demonstrated that only Y75W mutation of these three variants essentially influenced the functional activity of ArdA: the antimodification activity was restored to approximately 90-99%. It is assumed that R64 ArdA inhibits formation of the complex between unmodified DNA and the R subunit of the type I restriction-modification enzyme EcoKI (R2M2S), which translocates and cleaves DNA. ColIb-P9 ArdA protein is capable of forming the DNA complex not only with the R subunit, but also with the S subunit, which contacts sK site (containing modified adenine residues) in DNA. ArdA bound to the specific sK site inhibits concurrently the endonuclease and methylase activities of EcoKI (R2M2S), while ArdA bound to the nonspecific site in the R subunit blocks only its endonuclease activity.

The DNA-mimic antirestriction proteins ArdA ColIB-P9, Arn T4, and Ocr T7 as activators of H-NS-dependent gene transcription.

The antirestriction proteins ArdA ColIb-P9, Arn T4 and Ocr T7 specifically inhibit type I and type IV restriction enzymes and belong to the family of DNA-mimic proteins because their three-dimensional structure is similar to the double-helical B-form DNA. It is proposed that the DNA-mimic proteins are able to bind nucleoid protein H-NS and alleviate H-NS-silencing of the transcription of bacterial genes. Escherichia coli lux biosensors were constructed by inserting H-NS-dependent promoters into a vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE operon. It was demonstrated that the DNA-mimic proteins ArdA, Arn and Ocr activate the transcription of H-NS-dependent promoters of the lux operon of marine luminescent bacteria (mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio logei), and the dps gene from E. coli. It was also demonstrated that the ArdA antirestriction protein, the genes of which are located on transmissive plasmids ColIb-P9, R64, PK101, decreases levels of H-NS silencing of the PluxC promoter during conjugation in the recipient bacteria.

p53 and recombination intermediates: role of tetramerization at DNA junctions in complex formation and exonucleolytic degradation.

Heteroduplex joints represent intermediates of Rad51-dependent recombination processes, which are recognized by p53 with extremely high affinities, in a manner independent of the DNA sequence content. To determine the structural elements required for complex formation, we monitored DNA-binding by protection against restriction endonuclease cleavage. We show that wild-type (wt) p53 interacts with heteroduplex joints in the proximity of the flexible junction. Association of p53 within this junction region was also observed with preformed Rad51-heteroduplex complexes, whereas SSB counteracted p53 binding. At a distance of 31 bp from the junction p53 established very few contacts with the heteroduplex, despite the presence of an A-G mismatch. Consistently, p53-dependent exonucleolytic degradation decreased when we raised the distance between the junction and the heteroduplex terminus by 27 bp. Different from the cancer-related mutant p53(273H), which did not recognize the junction, tetramerization defective p53-1262 was protection competent but displayed reduced complex stability in gel shifts. Moreover, p53-1262 performed exonucleolytic activities towards ssDNA like wtp53, but reduced degradation of heteroduplex joints. These results suggest that during recombination wild-type p53, as a tetramer, stably binds to strand transfer regions, enabling the protein to exonucleolytically correct heteroduplex intermediates early after strand invasion.

Differential inhibition of a restriction enzyme by quinoxaline antibiotics.

The inhibition of cleavage by HpaI at two well-defined restriction sites in linearised phi X174-RF DNA by quinoxaline antibiotics has been investigated. Echinomycin, which displays a certain preference for binding to GC basepairs, inhibits cleavage at one site much more than the other, whereas triostin A, which displays less pronounced sequence-selectivity, inhibits both sites about equally. Other congeners inhibit reaction at the two sites with varying effectiveness. The results demonstrate the usefulness of studying inhibition of cleavage at specific sites by restriction enzymes as a means of exploring the specificity of DNA-ligand interactions.

Role of the reactive cysteine residue in restriction endonuclease Cfr9I.

Chemical modification studies were performed to elucidate the role of Cys-residues in the catalysis/binding of restriction endonuclease Cfr9I. Incubation of restriction endonuclease Cfr9I with N-ethylmaleimide (NEM), iodoacetate, 5,5'-dithiobis (2-nitrobenzoic acid) at pH 7.5 led to a complete loss of the catalytic activity. However, no enzyme inactivation was detectable after modification of the enzyme with iodoacetamide and methyl methanethiosulfonate. Complete protection of the enzyme against inactivation by NEM was observed in the presence of substrate implying that Cys-residues may be located at or in the vicinity of the active site of enzyme. Direct substrate-binding studies of native and modified restriction endonuclease Cfr9I using a gel-mobility shift assay indicated that the modification of the enzyme by NEM was hindered by substrate binding. A single Cys-residue was modified during the titration of the enzyme with DTNB with concomitant loss of the catalytic activity. The pH-dependence of inactivation of Cfr9I by NEM revealed the modification of the residue with the pKa value of 8.9 +/- 0.2. The dependence of the reaction rate of substrate hydrolysis by Cfr9I versus pH revealed two essential residues with pKa values of 6.3 +/- 0.15 and 8.7 +/- 0.15, respectively. The evidence presented suggests that the restriction endonuclease Cfr9I contains a reactive sulfhydryl residue which is non-essential for catalysis, but is located at or near the substrate binding site.

Inhibition of the type I restriction-modification enzymes EcoB and EcoK by the gene 0.3 protein of bacteriophage T7.

The gene 0.3 protein of bacteriophage T7 is a potent inhibitor of the restriction-modification enzymes EcoB and EcoK, both in vivo and in vitro. We have analyzed the ability of purified 0.3 protein to inhibit different steps in the reactions of EcoB and EcoK with DNA. Most of our experiments were done with EcoK, but selected tests with EcoB indicate that the two enzymes are affected by 0.3 protein in the same way. Purified 0.3 protein binds tightly to free enzyme, apparently to one of the small subunits, and prevents it from binding to DNA. If EcoK is allowed to form specific recognition complexes with unmodified DNA before 0.3 protein is added, relatively low levels of 0.3 protein prevent the nuclease activity that would otherwise appear upon addition of ATP, but considerably higher levels are needed to prevent formation of filter-binding complexes or ATPase activity. This, together with other results, suggests that the binding site for 0.3 protein is protected in recognition complexes and in the early stages of the ATP-stimulated reactions, but that it becomes accessible again before cleavage of the DNA, perhaps after the translocation step. If added after the nuclease reaction is substantially over, 0.3 protein has little effect on ATPase activity, and indeed, the subunit having the binding site for 0.3 protein apparently dissociates from the enzyme-DNA complex. The methylase activity of EcoK on hemi-methylated recognition sites is strongly inhibited by 0.3 protein added at any stage of the reaction.

Peptide inhibitors of DNA cleavage by tyrosine recombinases and topoisomerases.

The study of biochemical pathways requires the isolation and characterization of each and every intermediate in the pathway. For the site-specific recombination reactions catalyzed by the bacteriophage lambda tyrosine recombinase integrase (Int), this has been difficult because of the high level of efficiency of the reaction, the highly reversible nature of certain reaction steps, and the lack of requirements for high-energy cofactors or metals. By screening synthetic peptide combinatorial libraries, we have identified two related hexapeptides, KWWCRW and KWWWRW, that block the strand-cleavage activity of Int but not the assembly of higher-order intermediates. Although the peptides bind DNA, their inhibitory activity appears to be more specifically targeted to the Int-substrate complex, insofar as inhibition is resistant to high levels of non-specific competitor DNA and the peptides have higher levels of affinity for the Int-DNA substrate complex than for DNA alone. The peptides inhibit the four pathways of Int-mediated recombination with different potencies, suggesting that the interactions of the Int enzyme with its DNA substrates differs among pathways. The KWWCRW and KWWWRW peptides also inhibit vaccinia virus topoisomerase, a type IB enzyme, which is mechanistically and structurally related to Int. The peptides differentially affect the forward and reverse DNA transesterification steps of the vaccinia topoisomerase. They block formation of the covalent vaccinia topoisomerase-DNA intermediate, but have no apparent effect on DNA religation by preformed covalent complexes. The peptides also inhibit Escherichia coli topoisomerase I, a type IA enzyme. Finally, the peptides inhibit the bacteriophage T4 type II topoisomerase and several restriction enzymes with 2000-fold lower potency than they inhibit integrase in the bent-L pathway.

Ethidium-bromide-inhibited restriction endonucleases cleave one strand of circular DNA.

We analyzed the effect of ethidium bromide (EtBr) on the cleavage of closed circular pBR322 DNA molecules by six restriction enzymes which make staggered or flush cuts (EcoRI, HindIII, BglI, PstI, HincII, PvuII). EtBr concentrations and reaction temperatures were determined at which DNA molecules with single-strand breaks were the major reaction product of digestion by all the enzymes. However, the amounts of intermediates which could be isolated differed for various enzymes. The results extend previous studies, showing that sequential cleavage of the DNA strands probably is a general property of restriction endonucleases.

Distamycin A and its analogs as agents for blocking of endo R. EcoRI activity.

Distamycin A (Dst) and its analogs protect the lambda phage DNA from cleavage with endoR. EcoRI and show selective affinity for different recognition sites of endoR. EcoRI on this DNA producing enlarged DNA fragments of various composition and length. The affinity of the antibiotic for DNA is influenced by the number of pyrrol carboxamide units in Dst molecule and does not strongly depend on the substitution of the N-methyl group by the N-propyl one. Since in the complex with DNA the antibiotics of the Dst type are localized in its minor groove a conclusion can be made that the minor groove of DNA is needed for the interaction of the restriction endonuclease with DNA.

EcoRI activity: enzyme modification or activation of accompanying endonuclease?

A study has been made of the factors and mechanism leading to appearance of the so-called EcoRI activity described by Polisky et al. (1975) in the restrictase EcoRI preparations. The preparations of purified restrictase EcoRI, precipitated at 0.9 ammonium sulphate saturation, as well as that obtained using standard techniques have been found to contain an admixture of an endonuclease which at neutral pH and high ionic strength multiply cleaves those DNAs which normally have only one recognition site for EcoRI. Under the standard conditions for EcoRI digestion this activity is found only when large amounts of freshly isolated enzyme are added to the incubation mixture and it is sharply enhanced by replacement of Mg2+ with Mn2+. The number and size of DNA fragments produced under such conditions practically do not differ from those found under the so-called EcoRI conditions, that is for alkaline pH values and low ionic strength. The optimum incubation mixture for the EcoRI activity has been found to be 10 mM Tris . HCl buffer (pH 8.8) + 2 mM Mn2+. Similar activity is induced also by addition to EcoRI solution of 40--50% glycerol or a number of organic solvents (dimethylacetamide (DMA), dimethylformamide (DMF), dimethylsulphoxide (DMSO), sulphalane (SP) in concentrations from 1 to 6%. The EcoRI activity induced by 50% glycerol or at alkaline pH values and low ionic strength is suppressed or sharply inhibited by 2--3 mM parachloromercuribenzoate (PCMB), while EcoRI is not sensitive to this agent. The DNA fragments cleaved by EcoRI have cohesive termini and can be easily ligated. It is suggested that the EcoRI activity can be due not only (or largely not) to modification of the "recognizing capacity" of the EcoRI restrictase but not activation of a latent specific endonuclease which is present in the restrictase preparation as an impurity.