Double-stranded DNA Cleavage Research Articles

DNA Cleavage of a Cryptic Recombination Signal Sequence by RAG1 and RAG2: IMPLICATIONS FOR PARTIAL VH GENE REPLACEMENT

Antibody and T cell receptor genes are assembled from gene segments by V(D)J recombination to produce an almost infinitely diverse repertoire of antigen specificities. Recombination is initiated by cleavage of conserved recombination signal sequences (RSS) by RAG1 and RAG2 during lymphocyte development. Recent evidence demonstrates that recombination can occur at noncanonical RSS sites within Ig genes or at other loci, outside the context of normal lymphocyte receptor gene rearrangement. We have characterized the ability of the RAG proteins to bind and cleave a cryptic RSS (cRSS) located within an Ig V(H) gene segment. The RAG proteins bound with sequence specificity to either the consensus RSS or the cRSS. The RAG proteins nick the cRSS on both the top and bottom strands, thereby bypassing the formation of the DNA hairpin intermediate observed in RAG cleavage of canonical RSS substrates. We propose that the RAG proteins may utilize an alternative mechanism for double-stranded DNA cleavage, depending on the substrate sequence. These results have implications for further diversification of the antigen receptor repertoire as well as the role of the RAG proteins in genomic instability.

Structures, spectra, and DNA-binding properties of mixed ligand copper(II) complexes of iminodiacetic acid: The novel role of diimine co-ligands on DNA conformation and hydrolytic and oxidative double strand DNA cleavage

The coordination geometry around copper(II) in [Cu(imda)(phen)(H 2O)] ( 1) (H 2imda = iminodiacetic acid, phen = 1,10-phenanthroline) is described as distorted octahedral while those in [Cu(imda)(5,6-dmp)] ( 2) (5,6-dmp = 5,6-dimethyl-1,10-phenanthroline) and [Cu(imda)(dpq)] ( 3) (dpq = dipyrido-[3,2- d:2′,3′- f]-quinoxaline) as trigonal bipyramidal distorted square-based pyramidal with the imda anion facially coordinated to copper(II). Absorption spectral ( K b: 1, 0.60 ± 0.04 × 10 3; 2, 3.9 ± 0.3 × 10 3; 3, 1.7 ± 0.5 × 10 4 M −1) and thermal denaturation studies (Δ T m: 1, 5.70 ± 0.05; 2, 5.5 ± 10; 3, 10.6 ± 10 °C) and viscosity measurements indicate that 3 interacts with calf thymus DNA more strongly than 1 and 2. The relative viscosities of DNA bound to 1 and 3 increase while that of DNA bound to 2 decreases indicating formation of kinks or bends and/or conversion of B to A conformation as revealed by the decrease in intensity of the helicity band in the circular dichroism spectrum of DNA. While 1 and 3 are bound to DNA through partial intercalation, respectively, of phen ring and the extended planar ring of dpq with DNA base stack, the complex 2 is involved in groove binding. All the complexes show cleavage of pBR322 supercoiled DNA in the presence of ascorbic acid with the cleavage efficiency varying in the order 3 > 1 > 2. The highest oxidative DNA cleavage of dpq complex is ascribed to its highest Cu(II)/Cu(I) redox potential. Oxidative cleavage studies using distamycin reveal minor groove binding for the dpq complex but a major groove binding for the phen and 5,6-dmp complexes. Also, all the complexes show hydrolytic DNA cleavage activity in the absence of light or a reducing agent with cleavage efficiency varying in the order 1 > 3 > 2.

Double-strand DNA cleavage by copper complexes of 2,2′-dipyridyl with electropositive pendants

Two highly charged cationic copper(II) complexes have been synthesized and characterized structurally and spectroscopically: [Cu(L1)2(Br)](ClO4)5 (1) and [Cu(L2)2(Br)](ClO4)5 (2) (L1= 5,5'-di(1-(triethylammonio)methyl)-2,2'-dipyridyl cation and L2= 5,5'-di(1-(tributylammonio)methyl)-2,2'-dipyridyl cation bidentate ligands). X-Ray structures show that Cu(II) ions in both complexes have a trigonal-bipyramidal CuN4Br-configuration. Two nitrogen atoms of the electropositive pendants and coordinated bromine atom basically array in a straight line. Their close distances of N[dot dot dot]Br atoms are 5.772 and 5.594 A, respectively, which is comparable to that of adjacent phosphodiesters in B-form DNA (ca. 6 A). In the absence of reducing agent, supercoiled plasmid DNA cleavage by the complexes has been performed and their hydrolytic mechanisms have been investigated. The pseudo-Michaelis-Menten kinetic parameters (kcat), 4.15 h(-1) for 1, 0.43 h(-1) for 2 and 0.61 h(-1) for [Cu(bipy)(NO3)2], were obtained. This result indicates that 1 exhibits markedly higher nuclease activity than its corresponding analogues. The high ability of DNA cleavage for 1 is attributed to the effective cooperation of the metal moiety and two positive pendants since the array of linear tri-binding sites matches with one of three phosphodiester backbones of nucleic acid.

Mva1269I: A Monomeric Type IIS Restriction Endonuclease from Micrococcus Varians with Two EcoRI- and FokI-like Catalytic Domains

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5'-GAATGCN / -3'/5'-NG / CATTC-3' and cuts top and bottom DNA strands at positions, indicated by the "/" symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two "PD-(D/E)XK" domains. The N-terminal domain is related to the 5'-GAATTC-3'-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites.

Impact of the C-Terminal Domain of Topoisomerase IIα on the DNA Cleavage Activity of the Human Enzyme

The enzymatic function of the C-terminal domain of eukaryotic topoisomerase II is not well defined. This region of the enzyme is highly variable and hydrophilic and contains nuclear localization signals and phosphorylation sites. In contrast to eukaryotic topoisomerase II, type II enzymes from chlorella virus completely lack the C-terminal domain. These viral enzymes are characterized by a robust DNA cleavage activity, high coordination between their two active site tyrosyl residues, and reduced sensitivity to anticancer drugs. As a first step toward characterizing the contribution of the C-terminal domain of human topoisomerase IIalpha to enzyme function, the protein was truncated at amino acid 1175, which corresponds to the C-terminal residue of Paramecium bursaria chlorella virus-1 topoisomerase II as determined by BLAST sequence alignment. Although the overall catalytic activity of the resulting enzyme, hTop2alphaDelta1175, was lower than that of full-length topoisomerase IIalpha, the mutant protein displayed a double-stranded DNA cleavage activity that was approximately 2-3-fold higher. While the DNA breaks created by hTop2alphaDelta1175 were primarily double stranded, cuts generated by topoisomerase IIalpha were primarily single stranded. Thus, the enhanced cleavage observed for hTop2alphaDelta1175 appears to be due, at least in part, to an increase in active site coordination. Finally, hTop2alphaDelta1175 displayed a distinctly lower susceptibility to anticancer agents than did topoisomerase IIalpha, despite the fact that it showed a similar binding affinity for etoposide. Therefore, the C-terminal domain of human topoisomerase IIalpha appears to play significant roles in modulating the DNA cleavage/ligation reaction of the enzyme and its response to anticancer agents.

The 5S rRNA maturase, ribonuclease M5, is a Toprim domain family member

The maturation of 5S ribosomal RNA in low G+C Gram-positive bacteria is catalyzed by a highly conserved, ∼190 residue, enzyme, called ribonuclease M5 (RNase M5). Sequence alignment had predicted that the N-terminal half of RNase M5 would consist of a Toprim domain, a protein fold found in type IA and type II topoisomerases, DnaG-like primases, OLD family nucleases and RecR proteins [L. Aravind, D. D. Leipe and E. V. Koonin (1998) Nucleic Acids Res., 26, 4205–4213]. Here, we present structural modelling data and a mutational analysis of RNase M5 that confirms this hypothesis. The N-terminal half of RNase M5 can be fitted to the Toprim domain of the DnaG catalytic core. Mutation of amino acid residues highly conserved among RNase M5 enzymes and members of the Toprim domain family showed that alteration of residues critical for topoisomerase and primase activity also had a dramatic effect on the cleavage of 5S rRNA precursor by RNase M5 both in vivo and in vitro. This suggests that the mechanisms of double-stranded RNA cleavage by RNase M5 and double-stranded DNA cleavage by members of the topoisomerase family are related.

Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage

Custom-designed zinc finger nucleases (ZFNs) are becoming powerful tools in gene targeting—the process of replacing a gene within a genome by homologous recombination. Here, we have studied the DNA cleavage by one such ZFN, ΔQNK-FN, in order to gain insight into how ZFNs cleave DNA and how two inverted sites promote double-strand cleavage. DNA cleavage by ΔQNK-FN is greatly facilitated when two ΔQNK-binding sites are close together in an inverted orientation. Substrate cleavage was not first order with respect to the concentration of ΔQNK-FN, indicating that double-strand cleavage requires dimerization of the FokI cleavage domain. Rates of DNA cleavage decrease as the substrate concentrations increase, suggesting that the ΔQNK-FN molecules are effectively “trapped” in a 1:1 complex on DNA when the DNA is in excess. The physical association of two ZFN monomers on DNA was monitored by using the biotin-pull-down assay, which showed that the formation of ΔQNK-FN active complex required both binding of the two ΔQNK-FN molecules to specific DNA sites and divalent metal ions.

MnlI—The member of H-N-H subtype of Type IIS restriction endonucleases

The Type IIS restriction endonuclease MnlI recognizes the non-palindromic nucleotide sequence 5′-CCTC(N)7/6↓ and cleaves DNA strands as indicated by the arrow. The genes encoding MnlI restriction–modification system were cloned and sequenced. It comprises N6-methyladenine and C5-methylcytosine methyltransferases and the restriction endonuclease. Biochemical studies revealed that MnlI restriction endonuclease cleaves double- and single-stranded DNA, and that it prefers different metal ions for hydrolysis of these substrates. Mg 2+ ions were shown to be required for the specific cleavage of double-stranded DNA, whereas Ni 2+ and some other transition metal ions were preferred for nonspecific cleavage of single-stranded DNA. The C-terminal part of MnlI restriction endonuclease revealed an intriguing similarity with the H-N-H type nucleolytic domain of bacterial toxins, Colicin E7 and Colicin E9. Alanine replacements in the conserved sequence motif 306Rx 3ExHHx 14Nx 8H greatly reduced specific activity of MnlI, and some mutations even completely inactivated the enzyme. However, none of these mutations had effect on MnlI binding to the specific DNA, and on its oligomerisation state as well. We interpret the presented experimental evidence as a suggestion that the motif 306Rx 3ExHHx 14Nx 8H represents the active site of MnlI. Consequentially, MnlI seems to be the member of Type IIS with the active site of the H-N-H type.

A new heterobinuclear FeIIICuII complex with a single terminal FeIII–O(phenolate) bond. Relevance to purple acid phosphatases and nucleases

A novel heterobinuclear mixed valence complex [Fe(III)Cu(II)(BPBPMP)(OAc)(2)]ClO(4), 1, with the unsymmetrical N(5)O(2) donor ligand 2-bis[{(2-pyridylmethyl)aminomethyl}-6-{(2-hydroxybenzyl)(2-pyridylmethyl)}aminomethyl]-4-methylphenol (H(2)BPBPMP) has been synthesized and characterized. A combination of data from mass spectrometry, potentiometric titrations, X-ray absorption and electron paramagnetic resonance spectroscopy, as well as kinetics measurements indicates that in ethanol/water solutions an [Fe(III)-(mu)OH-Cu(II)OH(2)](+) species is generated which is the likely catalyst for 2,4-bis(dinitrophenyl)phosphate and DNA hydrolysis. Insofar as the data are consistent with the presence of an Fe(III)-bound hydroxide acting as a nucleophile during catalysis, 1 presents a suitable mimic for the hydrolytic enzyme purple acid phosphatase. Notably, 1 is significantly more reactive than its isostructural homologues with different metal composition (Fe(III)M(II), where M(II) is Zn(II), Mn(II), Ni(II), or Fe(II)). Of particular interest is the observation that cleavage of double-stranded plasmid DNA occurs even at very low concentrations of 1 (2.5 microM), under physiological conditions (optimum pH of 7.0), with a rate enhancement of 2.7 x 10(7) over the uncatalyzed reaction. Thus, 1 is one of the most effective model complexes to date, mimicking the function of nucleases.

Lysine–enediyne conjugates as photochemically triggered DNA double-strand cleavage agents

Statistical analysis of DNA-photocleavage by two types of lysine-enediyne conjugates confirms that more double-strand breaks are produced than can be accounted for by coincident single-strand breaks.

Amplification of C1027-induced DNA cleavage and apoptosis by a quinacrine–netropsin hybrid molecule in tumor cell lines

We examined the effect of a newly synthesized DNA-binding ligand, quinacrine–netropsin hybrid molecule (QN), on cytotoxicity, apoptosis, and DNA strand breaks induced by an enediyne antitumor antibiotic, C1027. QN significantly enhanced C1027-induced cellular DNA strand breaks, caspase-3 activation, and DNA ladder formation, characteristic of apoptosis, in human HL-60 cells. Flow cytometry revealed that C1027-induced intracellular H 2O 2 generation was enhanced by QN, suggesting that QN enhances C1027-induced cytotoxic effect through H 2O 2-mediated apoptosis. QN also significantly enhanced C1027-induced apoptosis in BJAB cells, and the inhibition of apoptosis was observed in BJAB cells transfected with Bcl-2 gene. The experiment using 32P-labeled DNA fragments showed that the addition of QN enhanced C1027-induced double-stranded DNA cleavage at the 5′-AG G-3′/3′- TCC-5′ sequence (cutting sites are underlined). These results suggest that QN enhances C1027-induced antitumor effect via DNA cleavage and apoptosis. The present study shows a novel approach to the potentially effective anticancer therapy.

Synthesis, structure and properties of unsymmetrical μ-alkoxo-dicopper(II) complexes: biological relevance to phosphodiester and DNA cleavage and cytotoxic activity

The unsymmetric dinucleating ligand N-(2-hydroxybenzyl)- N, N′, N′-tris(2-pyridylmethyl)-1,3-diaminopropan-2-ol (L = H 2btppnol) and the corresponding copper(II) complex [Cu 2(Hbtppnol)(μ-CH 3COO)](ClO 4) 2 ( 1) have been recently reported in part in a short communication [Inorg. Chem. Commum. 8 (1999) 334]. In this study, we investigated the ability of complex 1 to promote the hydrolysis of P–O phosphate diester bonds in bis(2,4-dinitrophenyl) phosphate (2,4-BDNPP) and the cleavage of genomic and plasmid DNA molecules. Reaction of 1 with excess of the diester 2,4-BDNPP, at pH 7.0, results in the formation of the monoester phosphate coordinated [Cu 2(Hbtppnol)(μ-((NO 2) 2-C 6H 3)PO 4)]ClO 4 ( 3) complex, which was also characterized by X-ray crystallography. In addition, the stable μ-phosphate complex [Cu 2(Hbtppnol)(μ-(NO 2-C 6H 4)PO 4)](ClO 4) ( 2) obtained from the reaction of 4-nitrophenyl phosphate with complex 1 was also characterized by X-ray crystallography, indicating that 1 is unable to cleave monoester–phosphate bonds. The kinetics for the promotion of bis(2,4-dinitrophenyl) phosphate (2,4-BDNPP) hydrolysis by complex 1 was investigated as a function of pH, catalyst concentration and substrate concentration. On the basis of kinetic and potentiometric studies, the deuterium isotope effect ( k H/ k D ∼ 1) and the X-ray structure of the monoester phosphate coordinated [Cu 2(Hbtppnol)(μ-((NO 2) 2-C 6H 3)PO 4)]ClO 4 ( 3) complex as the product of the reaction, we demonstrated that the aquo/hydroxo [ Cu 2 II ( L ) ( OH 2 ) ( OH ) ] complex is the active species and the reaction occurs through the formation of a ternary complex in which one Cu II binds the substrate and the second copper center has a terminal bound hydroxide to attack the phosphorus atom, at physiological pH. A rate enhancement factor of ∼100 was calculated relative to that measured for the uncatalyzed reaction under identical conditions. Complex 1 effectively promotes the cleavage of double-stranded genomic and plasmid DNA, at physiological pH, probably through a hydrolytic mechanism in agreement with that proposed for the reaction of 1 with 2,4-BDNPP. Finally, cytotoxic activity of 1 in a human small cell lung carcinoma cell line (GLC4) and its cisplatin resistant subline (GLC4/CDDP) was studied and the IC 50 values were determined.

Studying the effect of a charged surface on the interaction of bleomycin with DNA using an atomic force microscope

The cleavage of DNA caused by the antitumoral drug bleomycin has been investigated using atomic force microscopy (AFM). This work deals with the effect that adsorbing DNA onto a positively- or negatively-charged surface has on the double-strand cleavage of DNA by Fe(III)/bleomycin. Quantitative analysis of the number of breaks per DNA molecule, in bulk and at the surface of the mica substrate, has been performed by analyzing AFM images. It turns out that the cleavage of DNA is strongly inhibited by a positively-charged surface. Our experiments can be interpreted using a simple electrostatic model. This paper is a first step in the study of DNA accessibility to ligand such as bleomycin, using AFM in liquids.

Synapsis of recombination signal sequences located in cis and DNA underwinding in V(D)J recombination.

V(D)J recombination requires binding and synapsis of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins, aided by a high-mobility group protein, HMG1 or HMG2. Double-strand DNA cleavage within this synaptic, or paired, complex is thought to involve DNA distortion or melting near the site of cleavage. Although V(D)J recombination normally occurs between RSSs located on the same DNA molecule (in cis), all previous studies that directly assessed RSS synapsis were performed with the two DNA substrates in trans. To overcome this limitation, we have developed a facilitated circularization assay using DNA substrates of reduced length to assess synapsis of RSSs in cis. We show that a 12/23 pair of RSSs is the preferred substrate for synapsis of cis RSSs and that the efficiency of pairing is dependent upon RAG1-RAG2 stoichiometry. Synapsis in cis occurs rapidly and is kinetically favored over synapsis of RSSs located in trans. This experimental system also allowed the generation of underwound DNA substrates containing pairs of RSSs in cis. Importantly, we found that the RAG proteins cleave such substrates substantially more efficiently than relaxed substrates and that underwinding may enhance RSS synapsis as well as RAG1/2-mediated catalysis. The energy stored in such underwound substrates may be used in the generation of DNA distortion and/or protein conformational changes needed for synapsis and cleavage. We propose that this unwinding is uniquely sensed during synapsis of an appropriate 12/23 pair of RSSs.

Spin trapping of 13C-labeled p-benzynes generated by Masamune-Bergman cyclization of bicyclic nine-membered enediynes.

Our understanding of the chemistry and biology of the enediyne class of antitumor antibiotics is still developing and faces further challenges. The cleavage of double-stranded DNA through hydrogen abstraction by enediyne-derived radicals is a generally well-accepted mechanism. However, the mechanisms of several unexpected and complex cytotoxic effects of enediynes,such as RNA and protein damage or the generation of p-quinone species, have yet to be fully elucidated. Despite several carefully crafted experimental and theoretical studies on simplified systems,the behavior of the major chemical perpetrators of such cytotoxicity, namely the biradical intermediates formed by natural enediynes, remains more a matter of conjecture than a point of fact.

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 approximately 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.

The carboxy-terminal portion of TnsC activates the Tn7 transposase through a specific interaction with TnsA.

Tn7 transposition requires the assembly of a nucleoprotein complex containing four self-encoded proteins, transposon ends, and target DNA. Within this complex, TnsC, the molecular switch that regulates transposition, and TnsA, one part of the transposase, interact directly. Here, we demonstrate that residues 504-555 of TnsC are responsible for TnsA/TnsC interaction. The crystal structure of the TnsA/TnsC(504-555) complex, resolved to 1.85 A, illustrates the burial of a large hydrophobic patch on the surface of TnsA. One consequence of sequestering this patch is a marked increase in the thermal stability of TnsA as shown by differential scanning calorimetry. A model based on the complex structure suggested that TnsA and a slightly longer version of the cocrystallized TnsC fragment (residues 495-555) might cooperate to bind DNA, a prediction confirmed using gel mobility shift assays. Donor DNA binding by the TnsA/TnsC(495-555) complex is correlated with the activation of the TnsAB transposase, as measured by double-stranded DNA cleavage assays, demonstrating the importance of the TnsA/TnsC interaction in affecting Tn7 transposition.

Double-strand hydrolysis of DNA by a magnesium(II) complex with diethylenetriamine.

The development of artificial nucleases that hydrolyze DNA or RNA is of great interest in molecular biology, biotechnology, and medicine. We now report that a magnesium(II) complex of diethylenetriamine (Mg-dien) can effectively promote the double-stranded cleavage of plasmid DNA and the dideoxynucleotide dApdA under physiological conditions of pH and temperature. Experiments performed in the presence of hydrogen peroxide, radical scavengers, or under rigorously anaerobic conditions indicate that DNA cleavage mediated by Mg-dien occurs via a hydrolytic path. Mg-dien efficiently hydrolyzes supercoiled pBR322 DNA and the pseudo-first-order rate constant at 37 degrees C and pH 8.0 is estimated to be 1.60 h(-1). The dinucleotide dApdA hydrolysis, with Mg-dien at 170 microM, shows a rate enhancement factor of ca. 5 x 10(8). 1H and 31P(1H) NMR studies show that Mg-dien effectively hydrolyzes 5'-dAMP to give deoxyadenosine and inorganic phosphate. While Mg2+ has been found at the catalytic sites of many natural nucleases, Mg-dien appears to be the first synthetic Mg2+-containing system capable of hydrolyzing dideoxynucleotides and DNA and thus may provide a simple model system to assist mechanistic studies of naturally occurring nucleases.

Resonance Raman studies of HOO-Co(III)bleomycin and Co(III)bleomycin: identification of two important vibrational modes, nu(Co-OOH) and nu(O-OH).

Bleomycin is an antitumor agent whose cytotoxicity is dependent on its ability to bind DNA in the nucleus and effect double-stranded DNA cleavage, which is difficult for the cell to repair. In order for this DNA cleavage to occur, bleomycin must, through a series of reactions, form a low-spin Fe(III) complex, the putative "activated" form of the drug, HOO-Fe(III)bleomycin. The relative strengths of the bonds in the Fe(III)-OOH linkage have not been determined due to the weakness of the hydroperoxo-to-iron(III) charge-transfer transition. The much more stable HOO-Co(III)bleomycin has often been studied as a structural analogue of HOO-Fe(III)bleomycin, and hence, an understanding of the relative bond strengths in the Co-OOH linkage may serve to enhance our understanding of the analogous Fe-OOH linkage. In this report, we present resonance Raman data that identify two important vibrational modes in the Co-OOH linkage, the stretching modes, nu(Co-OOH) and nu(O-OH). Both of these vibrational modes were found to be unperturbed by complexation of the drug with calf thymus DNA. Advantage was also taken of the isostructural realtionship between Fe-bleomycin and Co-bleomycin to analyze and assign the high-frequency modes for HOO-Co(III)bleomycin and Co(III)bleomycin (A(2) and B(2)). These data could be useful for future studies of photoactivated Co-bleomycin and Co-bleomycin analogues in an attempt to characterize oxygen-independent DNA damage pathways.

Engineering Strand-specific DNA Nicking Enzymes from the Type IIS Restriction Endonucleases BsaI, BsmBI, and BsmAI

More than 80 type IIA/IIS restriction endonucleases with different recognition specificities are now known. In contrast, only a limited number of strand-specific nicking endonucleases are currently available. To overcome this limitation, a novel genetic screening method was devised to convert type IIS restriction endonucleases into strand-specific nicking endonucleases. The genetic screen consisted of four steps: (1) random mutagenesis to create a plasmid library, each bearing an inactivated endonuclease gene; (2) restriction digestion of plasmids containing the wild-type and the mutagenized endonuclease gene; (3) back-crosses with the wild-type gene by ligation to the wild-type N-terminal or C-terminal fragment; (4) transformation of the ligated DNA into a pre-modified host and screening for nicking endonuclease activity in total cell culture or cell extracts of the transformants. Nt.BsaI and Nb.BsaI nicking endonucleases were isolated from BsaI using this genetic screen. In addition, site-directed mutagenesis was carried out to isolate BsaI nicking variants with minimal double-stranded DNA cleavage activity. The equivalent amino acid substitutions were introduced into BsmBI and BsmAI restriction endonucleases with similar recognition sequence and significant amino acid sequence identity and their nicking variants were successfully isolated. This work provides strong evidence that some type IIS restriction endonucleases carry two separate active sites. When one of the active sites is inactivated, the type IIS restriction endonuclease may nick only one strand.