Sequence preferences of DNA interstrand cross-linking agents: dG-to-dG cross-linking at 5'-CG by structurally simplified analogues of mitomycin C

The nucleotide sequence preferences of the DNA interstrand cross-linking agents dehydroretronecine diacetate (DHRA), 2,3-bis(acetoxymethyl)-1-methylpyrrole (BAMP), dehydromonocrotaline, and dehydroretrorsine were studied by using synthetic DNA duplex fragments and polyacrylamide gel electrophoresis (PAGE). These agents have structural features in common with the reductively activated aziridinomitosene of mitomycin C (MC). Like MC, they preferentially cross-linked DNA duplexes containing the duplex sequence 5'-CG. For DHRA and BAMP interstrand cross-linked DNA duplexes, PAGE analysis of iron(II)-EDTA fragmentation reactions revealed the interstrand cross-links to be deoxyguanosine to deoxyguanosine (dG-to-dG), again analogous to DNA cross-links caused by MC. Unlike MC, DHRA could be shown to dG-to-dG cross-link a 5'-GC sequence. Furthermore, the impact of flanking sequence on the efficiency of interstrand cross-linking at 5'-CG was reduced for BAMP, with 5'-TCGA and 5'-GCGC being equally efficiently cross-linked. Possible origins of the 5'-CG sequence recognition common to all of the agents are discussed. A model is presented in which the transition state for the conversion of monoadducts to cross-links more closely resembles ground-state DNA at 5'-CG sequences.     

Interaction of the antitumor antibiotic mitomycin C with Z-DNA

Mitomycin C (MC), an antitumor antibiotic, alkylated Z-DNAs such as poly(dG-dC)/Co(NH3)3+(6), poly(dG-m5dC)/Mg2+ and brominated poly(dG-dC) upon reductive activation. Computer-generated energy-minimized molecular models indicated that monofunctional alkylation of Z-DNA at the N2-position of guanine by MC did not distort Z-DNA geometry, but bifunctional alkylation, leading to interstrand crosslinks between two N2-positions of guanine was sterically unfavorable. The above three Z-DNA's were exposed both to monofunctionally and bifunctionally activated MC in separate experiments and the resulting covalent MC-polynucleotide complexes were examined for conformation and for covalent MC-adducts, by circular dichroism (CD) spectroscopy and HPLC analysis of nuclease digests, respectively. Monofunctionally activated MC alkylated all three polynucleotides in their Z-forms, resulting in the same monofunctional N2-guanine adduct as that known to be formed with B-DNA. Upon bifunctional activation of MC, poly(dG-dC/Co(NH3)3+(6) reverted to the B-form and bifunctional (cross-link) adducts were detected, identical again with those formed with B-DNA. Poly(dG-m5dC), however, remained in the Z-form after the alkylation and only a monofunctional adduct could be detected. It was concluded that Z-DNA is subject to monofunctional alkylation by MC but cannot be cross-linked. The latter process occurs only when the Z-DNA is labile enough [as is in the case of poly(dG-dC)] to have some B-form in equilibrium at the site of the first formed monolinked adduct; the cross-linking then occurs at such local B-sites, pulling the overall B in equilibrium Z equilibrium irreversibly to the left. These results are in accord with the predictions from the above modeling. The irreversible "lock" by the MC cross-link on B-DNA may be exploited for probing Z-DNA intermediacy in various DNA functions.     

Mechanism of monofunctional and bifunctional alkylation of DNA by mitomycin C

The relative amounts of monofunctional and bifunctional alkylation products of DNA with mitomycin C (MC) depend on whether one or both masked alkylating functions of MC are activated reductively; adduct 8 is the result of one function and adducts 7 and 9, formed as a pair, are the result of both functions being activated [Tomasz, M., Lipman, R., Chowdary, C., Pawlak, J., Verdine, G. L., & Nakanishi, K. (1987) Science (Washington, D.C.) 235, 1204-1208]. To determine the mechanism governing this differential reactivity of MC with DNA, MC-Micrococcus luteus DNA complexes formed under varying conditions in vitro were digested to nucleosides and adducts. Adduct distribution, analyzed by high-performance liquid chromatography, served as the measure of monofunctional and bifunctional activation. H2/PtO2 and xanthine oxidase/reduced nicotinamide adenine dinucleotide (NADH) activated MC mostly monofunctionally, and Na2S2O4 activated the drug bifunctionally under comparable conditions. Excess MC selectively suppressed, but excess PtO2 selectively promoted, bifunctional activation by H2/PtO2; excess xanthine oxidase and/or NADH also had promoting effects. O2 tested in the Na2S2O4 system was inhibitory. 10-Decarbamoyl-MC acted strictly monofunctionally under all conditions. Monoadducts bound to DNA were converted to bis adducts upon rereduction. A mechanism with the following features was derived: (i) Activation of MC at C-1 and C-10 is sequential (C-1 first). (ii) A one-time reduction is sufficient for both. (iii) Activation of the second function may be selectively inhibited by kinetic factors or O2. (iv) 7 and 9 are coproducts of bifunctional activation; their ratio depends on the DNA base sequence. (v) Activation of the second function involves an iminium intermediate. Direct applications to the action of MC in vivo are discussed.     

Duplex oligodeoxyribonucleotides cross-linked by mitomycin C at a single site: synthesis, properties, and cross-link reversibility

Oligodeoxyribonucleotides cross-linked by reductively activated mitomycin C (MC) were prepared and purified for the first time. The cross-linked products were structurally characterized by nucleoside and MC-nucleoside adduct analysis. Optimal conditions were established for the cross-linking reaction, resulting in high yields, typically in the 20-50% range. Nuclease digests of the cross-linked oligonucleotides yielded the same bifunctional MC-deoxyguanosine adduct as that previously isolated from DNA exposed to MC in vitro and in vivo [Tomasz et al. (1987) Science 235, 1204]. The cross-linked oligonucleotides displayed broad thermal melting profiles, greatly increased Tm, and complex circular dichroism spectra. Phosphodiester linkages at the cross-link were resistant to spleen exonuclease, nuclease P1, and TaqI and ClaI restriction endonucleases; snake venom diesterase action was uninhibited. The cross-links are stable to heat at neutral pH but are removed by treatment in hot piperidine or by the reducing agents Na2S2O4 and dithiothreitol. Mechanisms are proposed for these reactions. These studies define optimal methods for introducing mitomycin cross-links into DNA fragments at a specific site, providing a versatile tool to study the effects of the MC cross-links on DNA structure and function.     

Recognition between mitomycin C and specific DNA sequences for cross-link formation

An extensive series of oligodeoxyribonucleotides was reacted with reductively activated mitomycin C (MC), and the resulting cross-linked drug-oligonucleotide complexes were isolated by reverse-phase HPLC and characterized by nucleoside and MC-nucleoside adduct analysis. HPLC also served for assay of the yield of cross-linked oligonucleotides. AT-rich duplex oligonucleotides, containing a single central CG.CG, gave high yields of cross-links between the two guanines while those having GC.GC, instead, gave none. In another series, the central sequences CGC.GCG and CGC.ICG both yielded 50% cross-link while CGC.GCI was completely resistant. Cross-linking was conducted also in two steps: Oligonucleotides substituted monofunctionally by MC at guanine at either a CG or GC sequence were annealed with their complementary strands followed by reductive reactivation of the bound MC to form a cross-link. The CG oligomers were cross-linked quantitatively while the GC ones were again resistant. These results show unambiguously that the MC cross-link is absolutely specific to the CG.CG duplex sequence, confirming our previous finding [Chawla, A.K., Lipman, R., & Tomasz, M. (1987) in Structure and Expression, Volume 2: DNA and Its Drug Complexes (Sarma, R.H., & Sarma, M.H., Eds.) Adenine Press, Guilderland, NY]. Evidence is presented that this specificity is due to the specific orientation of the monofunctionally attached MC in the minor groove. Superimposed on the CG.CG requirement, a four-base-pair sequence preference was observed at PuCGPyr.PuCGPyr sequences. This suggests that the guanine N2 atom of GpPyr is more reactive toward the drug than that of GpPu, due to the favorable effect of the negative dipole of the O2 of the Pyr on the reaction; in accordance, GpT was more reactive than GpC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of the structural-energy aspects of complementary-addressed modifications of nucleic acid with reactive 4-]N-methyl-N-(2-chloroethyl)-amino]benzyl-3'- or 5'-phosphamide oligonucleotide derivatives by a molecular mechanics method

Calculations of probabilities of the complementary addressed modification of target NA by 3'- or 5'-reactive derivatives of oligonucleotides carrying a 4-[N-(2-chloroethyl)-N-methyl]aminobenzyl group attached to the 3'- or 5'-terminal phosphates through a phosphoroamide linkage have been made. It is shown that the structural basis of the high efficiency and positional specificity depending on the NA target base sequence is the extent of structural correspondence of the energetically optimal conformation of the active group in the complex to the mutual arrangement of the active group and nucleophilic site needed for the chemical reaction. The 3'-derivative has the highest dependence of efficiency and positional specificity of the alkylation on the target NA base sequence. The maximal positional specificity of the alkylation is found for the modification of the cytidine at the first position from the terminal complementary base pair at the 5'-end of the target NA. For the 5'-derivative, the alkylating ability was determined to depend on the insertion of additional methylene bridges into the standard phosphoroamide linker: two methylene groups provide for the maximal increase of the modification ability of the nucleophilic site of the target NA in the double-stranded part of the complex. The efficiency of alkylation of the target NA in a three component complex with oligonucleotide-effector also complementary to the target NA have been studied. It was found that formation of the three-component complex lead to an additional stabilization of the conformation needed for the reaction of the active group, in comparison with two-component complex, by means of the intercalation of the phenyl group of the reagent in the gap between the oligonucleotide derivative and the oligonucleotide effector.     

Rapid routes of synthesis of chemically reactive and highly radioactively labeled alpha- and beta-oligonucleotide derivatives for in vivo studies

Development of the antisense oligonucleotide strategy for the regulation of gene expression in vivo poses several problems: the stability of oligonucleotides toward intracellular nucleases, labeling of oligonucleotides with high specific radioactivity, improvements of penetration of oligonucleotides into living cells, and enhancement of antisense action by coupling of chemically active groups. In the present paper synthesis of highly radioactively labeled [32P]- and [35S]oligonucleotide derivatives is described starting from both natural (beta) and nuclease-resistant (alpha) anomers of oligonucleotides. Conditions for preparative phosphorylation and thiophosphorylation suitable for oligonucleotides of various lengths, base composition, and anomeric forms were established. The stability of the phosphoramide bond under in vivo experimental conditions was checked. The methods of terminal phosphate chemical activation and terminal thiophosphate alkylation were applied to synthesize oligonucleotides equipped with hydrophobic, intercalating, alkylating, and photoactivatable groups. In the case of porphyrin-oligonucleotide conjugates, a series of new monofunctional porphyrin derivatives bearing a free aliphatic amino group was developed.     

Reductive alkylation of DNA by mitomycin A, a mitomycin with high redox potential

The mitomycins are a group of antitumor antibiotics that covalently bind to DNA upon reductive activation. Mitomycin A (1b; MA) is more toxic than its clinically useful mitomycin C (1a; MC). The greater toxicity of mitomycin A has been previously attributed to its higher reduction potential. In this report, the DNA alkylation products of reductively activated MA were isolated and characterized by conversion to the known 7-amino mitosene-deoxyguanosine adducts. The three major adducts formed were identified as a monoadduct, N2-(2"beta-amino-7"-methoxymitosen-1"alpha-yl)- 2'-deoxyguanosine (5), a decarbamoyl monoadduct, N2-(2"beta-amino-10"-decarbamoyl-7"-methoxymitosen-1"alpha-y l)-2'- deoxyguanosine (6), and a bisadduct, N2-(2"beta-amino-10"-deoxyguanosin-N2-yl-7-methoxymitosen-1" alpha- yl)-2'-deoxyguanosine (7). Under all reductive activation conditions employed, MA selectively alkylated the 2-amino group of guanine in DNA, like MC. In addition, both MA and MC alkylated DNA and cross-linked oligonucleotides to a similar extent. However, variations in the reductive activation conditions (H2/PtO2, Na2S2O4, or enzymatic) affected the distribution of the three major MA adducts in a different manner than the distribution of MC adducts was affected. A mechanism is proposed wherein the 7-methoxy substituent of MA allows initial indiscriminate activation of either of the drugs' two electrophilic sites. While oxygen inhibited cross-linking by MC, similar aerobic conditions exhibited little influence on the cross-linking ability of MA. Hence, the greater toxicity of MA may be influenced by increased and nonselective activation and cross-link formation in both aerobic and anaerobic cells. This effect is a direct consequence of the higher redox potential of MA as compared to MC.     

Solution structure of a guanine-N7-linked complex of the mitomycin C metabolite 2,7-diaminomitosene and DNA. Basis of sequence selectivity

2,7-Diaminomitosene (2,7-DAM), the major metabolite of the antitumor antibiotic mitomycin C, forms DNA adducts in tumor cells. 2,7-DAM was reacted with the deoxyoligonucleotide d(GTGGTATACCAC) under reductive alkylation conditions. The resulting DNA adduct was characterized as d(G-T-G-[M]G-T-A-T-A-C-C-A-C) (5), where [M]G stands for a covalently modified guanine, linked at its N7-position to C10 of the mitosene. The adducted oligonucleotide complements with itself, retaining 2-fold symmetry in the 2:1 drug-duplex complex, and provides well-resolved NMR spectra, amenable for structure determination. Adduction at the N7-position of G4 ([M]G, 4) is characterized by a downfield shift of the G4(H8) proton and separate resonances for G4(NH(2)) protons. We assigned the exchangeable and nonexchangeable proton resonances of the mitosene and the deoxyoligonucleotide in adduct duplex 5 and identified intermolecular proton-proton NOEs necessary for structural characterization. Molecular dynamics computations guided by 126 intramolecular and 48 intermolecular distance restraints were performed to define the solution structure of the 2,7-DAM-DNA complex 5. A total of 12 structures were computed which exhibited pairwise rmsd values in the 0.54-1.42 A range. The 2,7-DAM molecule is anchored in the major groove of DNA by its C10 covalently linked to G4(N7) and is oriented 3' to the adducted guanine. The presence of 2,7-DAM in the major groove does not alter the overall B-DNA helical structure. Alignment in the major groove is a novel feature of the complexation of 2,7-DAM with DNA; other known major groove alkylators such as aflatoxin, possessing aromatic structural elements, form intercalated complexes. Thermal stability properties of the 2,7-DAM-DNA complex 5 were characteristic of nonintercalating guanine-N7 alkylating agents. Marked sequence selectivity of the alkylation by 2,7-DAM was observed, using a series of oligonucleotides incorporating variations of the 5'-TGGN sequence as substrates. The selectivity correlated with the sequence specificity of the negative molecular electrostatic potential of the major groove, suggesting that the alkylation selectivity of 2,7-DAM is determined by sequence-specific variation of the reactivity of the DNA. The unusual, major groove-aligned structure of the adduct 5 may account for the low cytotoxicity of 2,7-DAM.     

Recognition of 5'-YpG-3' sequences by coupled stacking/hydrogen bonding interactions with amino acid residues

The combined biochemical and structural study of hundreds of protein-DNA complexes has indicated that sequence-specific interactions are mediated by two mechanisms termed direct and indirect readout. Direct readout involves direct interactions between the protein and base-specific atoms exposed in the major and minor grooves of DNA. For indirect readout, the protein recognizes DNA by sensing conformational variations in the structure dependent on nucleotide sequence, typically through interactions with the phosphodiester backbone. Based on our recent structure of Ndt80 bound to DNA in conjunction with a search of the existing PDB database, we propose a new method of sequence-specific recognition that utilizes both direct and indirect readout. In this mode, a single amino acid side-chain recognizes two consecutive base-pairs. The 3'-base is recognized by canonical direct readout, while the 5'-base is recognized through a variation of indirect readout, whereby the conformational flexibility of the particular dinucleotide step, namely a 5'-pyrimidine-purine-3' step, facilitates its recognition by the amino acid via cation-pi interactions. In most cases, this mode of DNA recognition helps explain the sequence specificity of the protein for its target DNA.     

Interstrand cross-linking reaction in triplexes containing a monofunctional transplatin-adduct.

Our aim was to determine whether a single transplatin monofunctional adduct, either trans-[Pt(NH3)2(dC)Cl]+ or trans-[Pt(NH3)2(dG)Cl]+ within a homopyrimidine oligonucleotide, could further react and form an interstrand cross-link once the platinated oligonucleotide was bound to the complementary duplex. The single monofunctional adduct was located at either the 5' end or in the middle of the platinated oligonucleotide. In all the triplexes, specific interstrand cross-links were formed between the platinated Hoogsteen strand and the complementary purine-rich strand. No interstrand cross-links were detected between the platinated oligonucleotides and non-complementary DNA. The yield and the rate of the cross-linking reaction depend upon the nature and location of the monofunctional adducts. Half-lives of the monofunctional adducts within the triplexes were in the range 2-6 h. The potential use of the platinated oligonucleotides to modulate gene expression is discussed.     

Catalytic diastereoselective sulfimidation of diaryl sulfides and application of chiral sulfimides to asymmetric allylic alkylation

The copper-catalyzed diastereoselective imidation of diaryl sulfides bearing a chiral oxazolinyl moiety at the ortho-position with [N-(p-toluenesulfonyl) imino]phenyliodinane (TsN=IPh) or Chloramine-T trihydrate [TsN(Cl)Na.3H2O] was successfully carried out to give the corresponding optically active N-tosylsulfimides in good yields. For example, the imidation of diphenyl sulfide bearing a methoxymethyl moiety at the 4-position of the oxazoline ring with TsN(Cl)Na.3H2O in acetonitrile in the presence of 10 mol% Cu(OTf)2 at 25 degrees C for 24 h affords the corresponding optically active N-tosylsulfimide in 52% isolated yield with a high diastereoselectivity of 99%. Hydrolysis of the optically active N-p-tosylsulfimides converts them into the corresponding optically active sulfimides in high yields without loss of diastereoselectivity. These novel optically active sulfimides and N-tosylsulfimides work as efficient chiral ligands for palladium(II)-catalyzed allylic alkylation of 1, 3-diphenyl-3-acetoxy-1-propene with dimethyl malonate to give the corresponding alkylation product quantitatively and with a high stereoselectivity (up to 90% ee).     

The sequence specificity of alkylation for a series of benzoic acid mustard and imidazole-containing distamycin analogues: the importance of local sequence conformation.

The covalent sequence specificity of a series of nitrogen mustard and imidazole-containing analogues of distamycin was determined using modified sequencing techniques. The analogues tether benzoic acid mustard (BAM) and possess either one, two or three imidazole units. Examination of the alkylation specificity revealed that BAM produced guanine-N7 lesions in a pattern similar to conventional nitrogen mustards. The monoimidazole-BAM conjugate also produced guanine-N7 alkylation in a similar pattern to BAM, but at a 100-fold lower dose. The diimidazole and triimidazole conjugates did not produce detectable guanine-N7 alkylation but only alkylated at selected sites in the minor groove. Unexpectedly, the alkylation specificity at equivalent doses was nearly identical to that found for the previously reported pyrrole-BAM conjugates. The consensus sequence, 5'-TTTTGPuwas strongly alkylated by the triimidazole conjugate in preference to other similar sites including three occurrences of 5'-TTTTAA. Footprinting studies were carried out to examine the non-covalent DNA binding interactions. These studies revealed that the tripyrrole- BAM conjugate bound non-covalently to the same AT-rich sites as distamycin. In contrast, whereas the Im3lexitropsin bound non-covalently to GC-rich sequences, the triimidazole-BAM conjugate did not detectably footprint to either GC- or AT-rich regions at equivalent doses. The results indicate that the alkylation event is not solely dictated by the non-covalent binding and might be influenced by a unique sequence dependent conformational feature of the consensus sequence 5'-TTTTGPu.     

Early inhibition of the Na+/K+-ATPase ion pump during acetaminophen-induced hepatotoxicity in rat

The status of Na+ regulation was examined during early stages of alkylation insult to rat liver. Na+/K+-ATPase activity in plasma membranes declined by 52% within 3 hr of treatment with 850 mg/kg acetaminophen. This loss preceded the release of alanine aminotransferase (2880 +/- 1550 U/ml) and necrosis (2+) seen at 24 hr. Activities of 5'-nucleotidase and Mg2+-ATPase and recovery of plasma membranes were comparatively unchanged at 3 hr. Because damage to Na+/K+-ATPase appeared early in the pathogenesis of acetaminophen hepatotoxicity, loss of hepatocellular Na+ regulation could represent one of the critical molecular consequences of lethal alkylation by acetaminophen.     

Instability of the monofunctional adducts in cis-[Pt(NH3)2(N7-N-methyl-2-diazapyrenium)Cl](2+)-modified DNA: rates of cross-linking reactions in cis-platinum-modified DNA.

Single- and double-stranded oligonucleotides containing a single monofunctional cis-[Pt(NH3)2(dG)(N7-N-methyl-2-diazapyrenium)]3+ adduct have been studied at two NaCl concentrations. In 50 mM and 1 M NaCl, the adducts within the single-stranded oligonucleotides are stable. In contrast, they are unstable within the corresponding double-stranded oligonucleotides. In 50 mM NaCl, the bonds between platinum and guanine or N-methyl-2,7-diazapyrenium residues are cleaved and subsequently, intra- or interstrand cross-links are formed as in the reaction between DNA and cis-DDP. In 1 M NaCl, the main reaction is the replacement of N-methyl-2,7-diazapyrenium residues by chloride which generates double-stranded oligonucleotides containing a single monofunctional cis-[Pt(NH3)2(dG)Cl]+ adduct. The rates of closure of these monofunctional adducts to bifunctional cross-links have been studied in 60 mM NaClO4. Within d(TG.CT/AGCA), d(CG.CT/AGCG) and d(AG.CT/AGCT) (the symbol.indicates the location of the adducts in the central sequences of oligonucleotides), the half-lifes (t1/2) of the cis-[Pt(NH3)2(dG)Cl]+ adducts are respectively 12, 6 and 2.8 hr and the cross-linking reactions occur between guanine residues on the opposite strands. Within d(AG.TC/GACT), d(CG.AT/ATCG) and d(TGTG./CACA) or d(TG.TG/CACA) t1/2 are respectively 1.6, 8 and larger than 20 hr and the intrastrand cross-links are formed at the d(AG), d(GA) and d(GTG) sites, respectively. The conclusion is that the rates of conversion of cis-platinum-DNA monofunctional adducts to minor bifunctional cross-links are dependent on base sequence. The potential use of the instability of cis-[Pt(NH3)2(dG)(N7-N-methyl-2-diazapyrenium)]3+ adducts is discussed in the context of the antisense strategy.     

Effect of phosphorothioate substitutions on DNA cleavage by Escherichia coli DNA topoisomerase I

To evaluate the structural influence of the DNA phosphate backbone on the activity of Escherichia coli DNA topoisomerase I, modified forms of oligonucleotide dA(7) were synthesized with a chiral phosphorothioate replacing the non-bridging oxygens at each position along the backbone. A deoxy-iodo-uracil replaced the 5'-base to crosslink the oligonucleotides by ultraviolet (UV) and assess binding affinity. At the scissile phosphate there was little effect on the cleavage rate. At the +1 phosphate, the rectus phosphorus (Rp)-thio-substitution reduced the rate of cleavage by a factor of 10. At the +3 and -2 positions from the scissile bond, the Rp-isomer was cleaved at a faster rate than the sinister phosphorus (Sp)-isomer. The results demonstrate the importance of backbone contacts between DNA substrate and E. coli topoisomerase I.     

Terminal nucleotide sequences of 17-S ribosomal RNA and its immediate precursor 18-S RNA in yeast.

The 5' and 3'-terminal nucleotide sequences of 17-S rRNA and its immediate precursor 18-S RNA from the yeast Saccharomyces carlsbergensis have been analysed. Identification of the terminal oligonucleotides, as present in Ti ribonuclease digests, was performed by diagonal procedures. The major (molar yield 0.9) 5'-terminal oligonucleotide (molar yield 0.15) with the overall composition pU (U2,C2)G was observed. 18-S precursor RNA was found to contain the same 5'-terminal sequences as 17-S rRNA. However, the 3'-terminal sequences of the two types of RNA appeared to be different. The 17-S rRNA yields the oligonucleotide A-U-C-A-U-U-AOH while at least half of the 18-S RNA molecules contain the sequence U-U-U-C-A-A-U-AOH. In addition 18-S RNA yields several minor 3'-terminal oligonucleotides which appear to be structurally related to the major 3'-terminal sequence. These results demonstrate that the extra nucleotides in 18-S RNA relative to 17-S RNA are located exclusively at the 3'-terminus of the 18-S RNA molecule. The possibility that the 3'-terminal nucleotide sequence of 18-S RNA plays a role in the maturation process is discussed.     

A complex family of class-II restriction endonucleases, DsaI-VI, in Dactylococcopsis salina

A series of class-II restriction endonucleases (ENases) was discovered in the halophilic, phototrophic, gas-vacuolated cyanobacterium Dactylococcopsis salina sp. nov. The six novel enzymes are characterized by the following recognition sequences and cut positions: 5'-C decreases CRYGG-3' (DsaI); 5'-GG decreases CC-3' (DsaII); 5'-R decreases GATCY-3' (DsaIII); 5'-G decreases GWCC-3' (DsaIV); 5'-decreases CCNGG-3' (DsaV); and 5'-GTMKAC-3' (DsaVI), where W = A or T, M = A or C, K = G or T, and N = A, G, C or T. In addition, traces of further possible activity were detected. DsaI has a novel sequence specificity and DsaV is an isoschizomer of ScrFI, but with a novel cut specificity. A purification procedure was established to separate all six ENases, resulting in their isolation free of contaminating nuclease activities. DsaI cleavage is influenced by N6-methyladenine residues [derived from the Escherichia coli-encoded DNA methyltransferase (MTase) M.Eco damI] within the overlapping sequence, 5'-CCRYMGGATC-3'; DsaV hydrolysis is inhibited by a C-5-methylcytosine residue in its recognition sequence (5'-CMCNGG-3'), generated in some DsaV sites by the E. coli-encoded MTase, M.Eco dcmI.