A role for the metal binding domain in determining the DNA sequence selectivity of Fe-bleomycin.

Previous studies of Fe-bleomycin-mediated DNA cleavage have established that the bithiazole moiety + C-terminal substituent of bleomycin are required for DNA binding, while the metal binding domain is responsible for O2 activation. Although recent studies have indicated that the metal binding domain also participates in DNA unwinding, and in determining the sequence and strand selectivity of DNA cleavage, no study has defined the structural domain that bears primary responsibility for the observed pattern of bleomycin-mediated DNA degradation. Presently, by the use of four synthetic analogs of bleomycin demethyl A2 having the functional domains connected by rigid spacers of varying lengths, the source of DNA cleavage specificity has been determined. When the four analogs cleaved 242- and 127-base pair 5'-32P-end-labeled DNA restriction fragments containing isolated Fe-bleomycin cleavage sites, all four produced cleavage at the same preferred sites. Because the (oligo)glycine spacers altered the distance between the domains by as much as 14 A, the identical cleavage patterns argue that the primary determinant of sequence specificity for these analogs is the metal binding domain.     

Some Properties of New DNA-Specific Bisbenzimidazole Fluorochromes without a Piperazine Ring

Three new bisbenzimidazole (BBI) compounds, which differ from Hoechst 33258 mainly by substitution of a N-dimethylaminopro-pylcarboxamide group in place of the N-methyl-piperazine ring, were studied for their DNA- and AT-base pair specificity as well as for their ability to be quenched by incorporated 5-bromodeoxy-uridine (BrdU). Each of them had DNA binding specificity comparable to or greater than that of Hoechst 33258 and each had a greater specificity for AT-rich regions than did Hoechst 33258. The dependence of fluorescence of new dyes on the BrdU-incorporation into DNA is different from that of Hoechst 33258 and related compounds with piperazine ring. The quenching effect is much weaker, and two of the new compounds (BBI-1 and BBI-2) even show somewhat enhanced binding (fluorescence) at lower concentrations. Certain BBI dyes without piperazine ring may have some advantage over Hoechst for accurate DNA [AT-specific] measurements. The piperazine ring appears to play an important role in the yet unknown mechanism of Hoechst quenching by incorporated BrdU.     

MamI, a novel class-II restriction endonuclease from Microbacterium ammoniaphilum recognizing 5'-GATNN decreases NNATC-3'

A new site-specific class-II restriction endonuclease, MamI, has been discovered in the nonsporulating Gram+ Microbacterium ammoniaphilum. MamI recognition sequence and cleavage positions were deduced using experimental and computer-assisted mapping and sequencing approaches. MamI cleavage specificity corresponds to: [formula: see text] The novel 43-kD enzyme recognizes a palindromic hexanucleotide interrupted by four ambiguous nucleotides. MamI cleavage positions are located in the center of the recognition sequence resulting in blunt-ended fragments after cleavage in the presence of Mg2+ ions. MamI is inhibited by N6-methyladenine residues. In case of overlapping sequences of MamI and Escherichia coli-coded DNA modification methyltransferase M.EcodamI (5'-[formula: see text]-3'), cleavage of DNA isolated from E. coli wild-type cells will be inhibited. By applying incubation conditions forcing star activity, relaxing of MamI sequence specificity is observed (MamI*).     

Netropsin, distamycin A, bis-netropsins as selective inhibitors of restrictases and DNAse I

The simultaneous analysis of DNAase I "footprinting" data and restriction endonucleases inhibition data was performed on the same DNA end-labelled fragment. The inhibition induced by netropsin, a number of bis-netropsins and distamycin A was investigated. These experiments led us to the following conclusions. The restriction endonucleases inhibition by the ligands is caused by the ligand molecules binding in the close vicinity to the restriction endonuclease recognition sequence. The zone of +/- 4 bp from the center of the restriction endonuclease recognition sequence can be defined as the zone of the influence of the bounded ligand on the restriction endonuclease. But in this case the intersection of recognition sequence and the binding site occupied by a single ligand molecule is not sufficient for the inhibition to occur. Restriction endonuclease cutting sites protected by netropsin can be predicted basing upon known nucleotide sequence specificity of netropsin. Netropsin and bis-netropsins show different nucleotide sequence specificity. This fact can be used for selective inhibition of restriction endonucleases.     

Activity and Specificity of the Bacterial PD-(D/E)XK Homing Endonuclease I-Ssp6803I

The restriction endonuclease fold [a three-layer α-β sandwich containing variations of the PD-(D/E)XK nuclease motif] has been greatly diversified during evolution, facilitating its use for many biological functions. Here we characterize DNA binding and cleavage by the PD-(D/E)XK homing endonuclease I-Ssp6803I. Unlike most restriction endonucleases harboring the same core fold, the specificity profile of this enzyme extends over a long (17 bp) target site. The DNA binding and cleavage specificity profiles of this enzyme were independently determined and found to be highly correlated. However, the DNA target sequence contains several positions where binding and cleavage activities are not tightly coupled: individual DNA base-pair substitutions at those positions that significantly decrease cleavage activity have minor effects on binding affinity. These changes in the DNA target sequence appear to correspond to substitutions that uniquely increase the free energy change between the ground state and the transition state, rather than simply decreasing the overall DNA binding affinity. The specificity of the enzyme reflects constraints on its host gene and limitations imposed by the enzyme's quaternary structure and illustrate the highly diverse repertoire of DNA recognition specificities that can be adopted by the related folds surrounding the PD-(D/E)XK nuclease motif.     

S-adenosyl-L-methionine is required for DNA cleavage by type III restriction enzymes.

The requirement of S-adenosyl-L-methionine (AdoMet) in the cleavage reaction carried out by type III restriction-modification enzymes has been investigated. We show that DNA restriction by EcoPI restriction enzyme does not take place in the absence of exogenously added AdoMet. Interestingly, the closely related EcoP15I enzyme has endogenously bound AdoMet and therefore does not require the addition of the cofactor for DNA cleavage. By employing a variety of AdoMet analogs, which differ structurally from AdoMet, this study demonstrates that the carboxyl group and any substitution at the epsilon carbon of methionine is absolutely essential for DNA cleavage. Such analogs could bring about the necessary conformational change(s) in the enzyme, which make the enzyme proficient in DNA cleavage. Our studies, which include native polyacrylamide gel electrophoresis, molecular size exclusion chromatography, UV, fluorescence and circular dichroism spectroscopy, clearly demonstrate that the holoenzyme and apoenzyme forms of EcoP15I restriction enzyme have different conformations. Furthermore, the Res and Mod subunits of the EcoP15I restriction enzyme can be separated by gel filtration chromatography in the presence of 2 M NaCl. Reconstitution experiments, which involve mixing of the isolated subunits, result in an apoenzyme form, which is restriction proficient in the presence of AdoMet. However, mixing the Res subunit with Mod subunit deficient in AdoMet binding does not result in a functional restriction enzyme. These observations are consistent with the fact that AdoMet is required for DNA cleavage. In vivo complementation of the defective mod allele with a wild-type mod allele showed that an active restriction enzyme could be formed. Furthermore, we show that while the purified c2-134 mutant restriction enzyme is unable to cleave DNA, the c2-440 mutant enzyme is able to cleave DNA albeit poorly. Taken together, these results suggest that AdoMet binding causes conformational changes in the restriction enzyme and is necessary to bring about DNA cleavage.     

The crystal structure of the rare-cutting restriction enzyme SdaI reveals unexpected domain architecture

Rare-cutting restriction enzymes are important tools in genome analysis. We report here the crystal structure of SdaI restriction endonuclease, which is specific for the 8 bp sequence CCTGCA/GG ("/" designates the cleavage site). Unlike orthodox Type IIP enzymes, which are single domain proteins, the SdaI monomer is composed of two structural domains. The N domain contains a classical winged helix-turn-helix (wHTH) DNA binding motif, while the C domain shows a typical restriction endonuclease fold. The active site of SdaI is located within the C domain and represents a variant of the canonical PD-(D/E)XK motif. SdaI determinants of sequence specificity are clustered on the recognition helix of the wHTH motif at the N domain. The modular architecture of SdaI, wherein one domain mediates DNA binding while the other domain is predicted to catalyze hydrolysis, distinguishes SdaI from previously characterized restriction enzymes interacting with symmetric recognition sequences.     

Pentamidine-induced alteration in restriction endonuclease cleavage of plasmid DNA

We have used restriction enzymes and DNaseI as probes to determine the specificity of pentamidine binding to plasmid DNA. Cleavage of plasmid pAZ130 by EcoRI, EcoRV and ApaI is inhibited by pentamidine, cleavage by XbaI, NotI and AvaI is unaffected, while cleavage by XhoI, which recognizes the same sequence as AvaI, is stimulated. DNaseI footprinting of DNA containing these restriction sites revealed that pentamidine protection is not strictly limited to AT-rich regions. We suggest that perturbation of the DNA micro- environment by pentamidine binding is responsible for its effect on nucleases.     

Distribution of topoisomerase II-mediated cleavage sites and relation to structural and functional landmarks in 830 kb of Drosophila DNA.

The pattern of sites for cleavage mediated by topoisomerase II was determined in 830 kb of cloned DNA from the Drosophila X chromosome, with the objectives of comparing it with mapped structural and functional landmarks and examining if the correlations with such landmarks reported in individual loci can be generalized to a region approximately 100 times longer. The relative frequencies of topoisomerase II cleavage sites in 247 restriction fragments from 67 clones were quantified by hybridization with probes prepared from DNA fragments which abutted all cleavage sites in each clone, selected through the covalently bound topoisomerase II subunit; the specificity and quantitative nature of this method were demonstrated using a plasmid DNA model. The 12 restriction fragments with strong nuclear scaffold attachment (SAR) activity, of which seven possess autonomous replication (ARS) activity, show statistically strong coincidence or contiguity ( P </=0.11) with regions of high topoisomerase II cleavage site frequency. These regions show no correlation with repetitive sequence or A/T or C/G content and some extend over >10 kb; their sensitivity is therefore unlikely to be due to alternating purine-pyrimidine repeats or regions of Z conformation, which are preferred motifs. The hypothesis that they possess intrinsic curvature is consistent with the similarity of their length and spacing to regions of predicted curvature in the 315 kb DNA of Saccharomyces cerevisiae chromosome III and with the reported strong binding preference of topoisomerase II for curved DNA. The topoisomerase II cleavage pattern in this DNA further shows that its relationships to functional properties seen in individual loci, especially to MAR/SAR and ARS activity and to the restricted accessibility of DNA to topoisomerase II in vivo, can be generalized to much longer regions of the genome.     

Inhibition of the restriction endonuclease BanII using modified DNA substrates. Determination of phosphate residues critical for the formation of an active enzyme-DNA complex

The restriction endonuclease BanII catalyzes the cleavage of double-stranded DNA and recognizes the degenerate sequence 5'-GPuGCPyC-3'. The poly-linker of M13mp18 contains one such sequence, 5'-GAGCTC-3'. The three other possible sites recognized by the enzyme were prepared by site-directed muta-genesis. The substitution of phosphate groups by phosphorothioate residues at some positions within the various recognition sites had relatively little effect on the rate of cleavage of the DNA. However, when the DNA contained a phosphorothioate group at the site of cleavage the rate of linearization of the DNA was decreased by a factor of 9. Interestingly, DNA which contained an additional phosphorothioate internucleotidic linkage immediately 3'-outside the recognition site could not be linearized by the enzyme. The results indicate that an important contact between enzyme and substrate is perturbed by the presence of the sulfur atom at this position.     

Cleavage of phosphorothioated DNA and methylated DNA by the type IV restriction endonuclease ScoMcrA

Many taxonomically diverse prokaryotes enzymatically modify their DNA by replacing a non-bridging oxygen with a sulfur atom at specific sequences. The biological implications of this DNA S-modification (phosphorothioation) were unknown. We observed that simultaneous expression of the dndA-E gene cluster from Streptomyces lividans 66, which is responsible for the DNA S-modification, and the putative Streptomyces coelicolor A(3)2 Type IV methyl-dependent restriction endonuclease ScoA3McrA (Sco4631) leads to cell death in the same host. A His-tagged derivative of ScoA3McrA cleaved S-modified DNA and also Dcm-methylated DNA in vitro near the respective modification sites. Double-strand cleavage occurred 16-28 nucleotides away from the phosphorothioate links. DNase I footprinting demonstrated binding of ScoA3McrA to the Dcm methylation site, but no clear binding could be detected at the S-modified site under cleavage conditions. This is the first report of in vitro endonuclease activity of a McrA homologue and also the first demonstration of an enzyme that specifically cleaves S-modified DNA.     

The SalGI restriction endonuclease. Enzyme specificity.

We have analysed the kinetics of DNA cleavage in the reaction between the SalGI restriction endonuclease and plasmid pMB9. This reaction is subject to competitive inhibition by DNA sequences outside the SalGI recognition site; we have determined the Km and Vmax. for the reaction of this enzyme at its recognition site and the KI for its interaction at other DNA sequences. We conclude that the specificity of DNA cleavage by the enzyme is only partly determined by the discrimination it shows for binding at its recognition sequence compared with binding to other DNA sequences.     

Determination of 5′-bromodeoxyuridine in DNA by buoyant density

Cesium chloride buoyant density analysis shows the density difference between the double-stranded alternating copolymers of dT and dA, poly(dAT·dAT) and of dA and BrdU, (dABU·dABU), to be 0.1730 g/cm³. A mammalian DNA fully substituted with 5′-bromodeoxyuridine (BrdU) increases in density over control, unsubstituted, DNA by a value within 1% of that predicted from analysis of the copolymers. A simple relationship is presented for the accurate determination of the extent of BrdU substitution in a DNA of heterogeneous base composition.     

Radiosensitization of Chinese hamster V79 cells by bromodeoxyuridine substitution of thymidine: enhancement of radiation-induced toxicity and DNA strand break production by monofilar and bifilar substitution

Bromodeoxyuridine (BrdU) competes with thymidine (TdR) for incorporation into DNA of exponentially growing V79-171 cells. Such cells show an enhancement of the radiation response as determined by clonogenic survival and DNA damage measured by filter elution techniques after doses up to 15 Gy. The degree of radiosensitization for both survival and rates of alkaline and neutral elution are dependent on percentage BrdU substitution and independent of whether BrdU is in one strand only (monofilar) or both strands (bifilar) of the DNA duplex: e.g., for 16% BrdU substitution distributed either monofilarly or partially bifilarly, there is an enhancement factor for Do of 1.55. At this percentage substitution, the enhancement factor for the rate of alkaline elution is 1.75 and that for the rate of neutral elution is 1.54. The greater the percentage BrdU substitution, the larger was the enhancement ratio for survival and radiation-induced strand breaks in both monofilarly and bifilarly substituted cells. The increase in cell radiosensitivity caused by BrdU substitution shows a better correlation with the increase in radiation-induced double-strand breaks than with the increase in radiation-induced single-strand breaks.     

A detailed study of the substrate specificity of a chimeric restriction enzyme.

Recently, the crystal structure of the designed zinc finger protein, DeltaQNK, bound to a preferred DNA sequence was reported. We have converted DeltaQNK into a novel site-specific endonuclease by linking it to the Fok I cleavage domain (FN). The substrate specificity and DNA cleavage properties of the resulting chimeric restriction enzyme (DeltaQNK-FN) were investigated, and the binding affinities of DeltaQNK and DeltaQNK-FN for various DNA substrates were determined. Substrates that are bound by DeltaQNK with high affinity are the same as those that are cleaved efficiently by DeltaQNK-FN. Substrates bound by DeltaQNK with lower affinity are cleaved with very low efficiency or not at all by DeltaQNK-FN. The binding of DeltaQNK-FN to each substrate was approximately 2-fold weaker than that for DeltaQNK. Thus, the fusion of the Fok I cleavage domain to the zinc finger motif does not change the DNA sequence specificity of the zinc finger protein and does not change its binding affinity significantly.     

Cleavage of adenine-modified functionalized DNA by type II restriction endonucleases

A set of 6 base-modified 2′-deoxyadenosine derivatives was incorporated to diverse DNA sequences by primer extension using Vent (exo-) polymerase and the influence of the modification on cleavage by diverse restriction endonucleases was studied. While 8-substituted (Br or methyl) adenine derivatives were well tolerated by the restriction enzymes and the corresponding sequences were cleaved, the presence of 7-substituted 7-deazaadenine in the recognition sequence resulted in blocking of cleavage by some enzymes depending on the nature and size of the 7-substituent. All sequences with modifications outside of the recognition sequence were perfectly cleaved by all the restriction enzymes. The results are useful both for protection of some sequences from cleavage and for manipulation of functionalized DNA by restriction cleavage.     

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies.

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.     

Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains

This study concerns chimeric restriction enzymes that are hybrids between a zinc finger DNA-binding domain and the non-specific DNA-cleavage domain from the natural restriction enzyme FokI. Because of the flexibility of DNA recognition by zinc fingers, these enzymes are potential tools for cleaving DNA at arbitrarily selected sequences. Efficient double-strand cleavage by the chimeric nucleases requires two binding sites in close proximity. When cuts were mapped on the DNA strands, it was found that they occur in pairs separated by ~4 bp with a 5′ overhang, as for native FokI. Furthermore, amino acid changes in the dimer interface of the cleavage domain abolished activity. These results reflect a requirement for dimerization of the cleavage domain. The dependence of cleavage efficiency on the distance between two inverted binding sites was determined and both upper and lower limits were defined. Two different zinc finger combinations binding to non-identical sites also supported specific cleavage. Molecular modeling was employed to gain insight into the precise location of the cut sites. These results define requirements for effective targets of chimeric nucleases and will guide the design of novel specificities for directed DNA cleavage in vitro and in vivo.     

Histones bind more tightly to bromodeoxyuridine-substituted DNA than to normal DNA.

Using a membrane filter assay, we have obtained results from both kinetic and competition experiments indicating that histones bind more strongly to bromodeoxyuridine-substituted DNA than to normal DNA. At 37 degrees C in our standard buffer of 0.2 M ionic strength, the rate of dissociation of histones H1, H2, and h4 from BrdU-substituted DNA is respectively 7, 4, and 2 times slower than it is from normal DNA. Competition experiments show an even greater difference between BrdU-substituted and normal DNA with respect to histone binding. The tighter binding of histones to BrdU-substituted DNA is of interest because of the known effects of BrdU on eukaryotic chromosome condensation and staining, virus induction, and the inhibition of differentiation.