Isolation and computer-aided characterization of MmeI, a type II restriction endonuclease from Methylophilus methylotrophus.

A Type II restriction endonuclease, MmeI, has been purified from the obligate methylotroph, Methylophilus methylotrophus. The enzyme was shown to have the non-palindromic recognition sequence 5'-T C C Pu A C (N)20-3', 3'-A G G Py T G (N)18-5' and to cleave (as indicated) on the 3' side, generating a two nucleotide 3' projection. Determination of the recognition sequence was achieved using two new computer programs; RECOG, which predicts recognition sequences from the pattern of restriction fragments obtained from DNAs of known sequence, and GELSIM, which generates graphical simulations of DNA band patterns obtained by gel electrophoresis of restriction digests of sequenced DNA molecules.     

Sequence-specific DNA cleavage by dipeptides disubstituted with chlorambucil and 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid.

Two dipeptides, each containing a lysyl residue, were disubstituted with chlorambucil (CLB) and 2,6-dimethoxyhydroquinone-3-mercaptoacetic acid (DMQ-MA): DMQ-MA-Lys(CLB)-Gly-NH2 (DM-KCG) and DMQ-MA-beta-Ala-Lys(CLB)-NH2 (DM-BKC). These peptide-drug conjugates were designed to investigate sequence-specificity of DNA cleavage directed by the proximity effect of the DNA cleavage chromophore (DMQ-MA) situated close to the alkylating agent (CLB) inside a dipeptide moiety. Agarose electrophoresis studies showed that DM-KCG and DM-BKC possess significant DNA nicking activity toward supercoiled DNA whereas CLB and its dipeptide conjugate Boc-Lys(CLB)-Gly-NH2 display little DNA nicking activity. ESR studies of DMQ-MA and DM-KCG both showed five hyperfine signals centered at g = 2.0052 and are assigned to four radical forms at equilibrium, which may give rise to a semiquinone radical responsible for DNA cleavage. Thermal cleavage studies at 90 degrees C on a 265-mer test DNA fragment showed that besides alkylation and cleavage at G residues, reactions with DM-KCG and DM-BKC show a preference for A residues with the sequence pattern: 5'-G-(A)n-Pur-3' > 5'-Pyr-(A)n-Pyr-3' (where n = 2-4). By contrast, DNA alkylation and cleavage by CLB occurs at most G and A residues with less sequence selectivity than seen with DM-KCG and DM-BKC. Thermal cleavage studies using N7-deazaG and N7-deazaA-substituted DNA showed that strong alkylation and cleavage at A residues by DM-KCG and DM-BKC is usually flanked on the 3' side by a G residue whereas strong cleavage at G residues is flanked by at least one purine residue on either the 5' or 3' side. At 65 degrees C, it is notable that the preferred DNA cleavage by DM-KCG and DM-BKC at A residues is significantly more marked than for G residues in the 265-mer DNA; the strongest sites of A-specific reaction occur within the sequences 5'-Pyr-(A)n-Pyr-3'; 5'-Pur-(A)n-G-3' and 5'-Pyr-(A)n-G-3'. In pG4 DNA, cleavage by DM-KCG and DM-BKC is much greater than that by CLB at room temperature and at 65 degrees C. It was also observed that DM-KCG and DM-BKC cleaved at certain pyrimidine residues: C40, T66, C32, T34, and C36. These cleavages were also sequence selective since the susceptible pyrimidine residues were flanked by two purine residues on both the 5' and 3' sides or by a guanine residue on the 5' side. These findings strongly support the proposal that once the drug molecule is positioned so as to permit alkylation by the CLB moiety, the DMQ-MA moiety is held close to the alkylation site, resulting in markedly enhanced sequence-specific cleavage.     

Nucleotide sequence of the 5'- and 3'- domains for rabbit 18S ribosomal RNA.

By direct RNA sequence analysis we have determined the primary structures of both the 5' and 3' domains for rabbit 18S ribosomal RNA. Purified 18S rRNA was labeled in vitro at either its 5' or 3' terminus with 32P, base-specifically fragmented enzymatically and chemically, and the resulting fragments electrophoretically fractionated by size in adjacent lanes of 140 cm long polyacrylamide sequencing gels run in 90% formamide. A phylogenetic comparison of both the mammalian 5' proximal 400 residues and the 3' distal 301 nucleotides with the previously determined yeast and Xenopus laevis 18S rRNA sequence shows extensive conservation interspersed with tracts having little homology. Clusters of G + C rich sequences are present within the mammalian 5' domain which are entirely absent in both the Xenopus laevis and yeast 18S rRNAs. Most base differences and insertions within the mammalian 18S rRNA when compared with yeast or Xenopus rRNA result in an increase in the G + C content of these regions. We have found nucleotide sequence analysis of the ribosomal RNA directly permits detection of both cistron heterogeneities and mapping of many of the modified bases.     

A restriction enzyme from Fusobacterium nucleatum 4H which recognizes GCNGC.

A site-specific restriction endonuclease Fnu4H I isolated from Fusobacterium nucleatum 4H recognizes the DNA nucleotide sequence 5'G C N G C-3'/3'-C G N C G-5' and cleaves as indicated by the arrows.     

Two new restriction endonucleases from Proteus vulgaris.

Two novel sequence-specific endonucleases have been isolated from Proteus vulgaris, ATCC 13315. PvuI recognizes the sequence: 5' C G A T decrease C G 3' 3' G C increase T A G C 5' and PvuII recognizes the sequence: 5' C A G decrease C T G 3' 3' G T C increase G A C 5' and cleave as indicated by the arrow (decrease). PvuI is an isoschizomer of XorII, RshI, and XniI. No enzyme with the specificity of PvuII has been described previously.     

Deuteriation of sugar protons simplify NMR assignments and structure determination of large oligonucleotide by the 1H-NMR window approach.

The concept of the 1H-NMR window has been developed and examined through a comparative study of NOESY spectra of a self-complementary Dickerson's dodecamer (I) [5'd(5C6G7C8G9A10A11T12T13C-14G15C16G)2(3')], a self-complementary 20-mer (II) [(5'd(1C2G3C4G5C6G7C8G9A10A11T12T13C14G15C16G17C18G19C20G)2(3')] in which the core part consists of the same Dickerson's dodecamer sequence with the flanking CGCG residues at both 3' and 5'-ends, and the partly-deuteriated (shown by underlined CGCG residues at both 3' and 5'-ends) analogous duplex (III) [5'd(1C2G3C4G5C6G7C8G9A10A11T12T13C14G15C16G17C18G19C20G)2(3')] in which the core 5C to 16G part (i.e. 1H-NMR window) consists of the natural Dickerson's dodecamer sequence. A comparison of their NOESY spectra clearly demonstrates that the severe overlap of proton resonances in the larger DNA duplex (II) has been successfully reduced in the partly-deuterated duplex (III) as a result of specific incorporations of the sugar-deuteriated nucleotide residues in the latter [stereospecific > 97 atom % 2H enrichment at H2', H2' and H3' sites, approximately 85 atom % 2H enrichment at H4' and approximately 20 atom % 2H enrichment at H1' (see refs. 10 and 11) in the 20-mer duplex (III)]. These simplifications of the resonance overlap by the deuteriation approach have enabled unequivocal chemical shift assignments and extraction of the quantitative NOE data in the 1H-NMR window part of duplex (III). A comparison of the 12-nucleotide long 1H-NMR window in (III) with that of the 12-mer duplex (I) also shows the scope of studying the changes in conformation and dynamics of the core 12-mer region in (III) which result from the increase of molecular weight due to the DNA chain extension. It is noteworthy that such a study is clearly impossible for the natural 20-mer (II) because of the inherent problem of the overlap of resonances.     

BspLS2I--a new site-specific endonuclease from the thermophilic bacteria Bacillus species LS2]

A new restriction endonuclease BspLS2I was isolated from the thermophilic bacterium Bacillus species LS2 and purified by blue sepharose and hydroxyapatite chromatographies. The enzyme is an isoschizomer of SduI from Streptococcus durans. BspLS2I recognizes the sequence 5' G(G/A/T)GC(C/T/A) decreases C 3' on double-stranded DNA and cleaves it is indicated by the arrow to yield sticky-ended DNA fragments. Maximum catalytic activity of endonuclease was found in 10 mM tris-HCl (pH 7.9) in the presence of 15-30 mM MgCl2 at 50 degrees C. The phage T4 glucosylated DNA is not cleaved by the enzyme.     

Thermodynamics of DNA duplexes with adjacent G.A mismatches.

The sequence 5'-d(ATGAGCGAAT) forms a very stable self-complementary duplex with four G.A mismatch base pairs (underlined) out of ten total base pairs [Li et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 26-30]. The conformation is in the general B-family and is stabilized by base-pair hydrogen bonding of an unusual type, by favorable base dipole orientations, and by extensive purine-purine stacking at the mismatched sites. We have synthesized 13 decamers with systematic variations in the sequence above to determine how the flanking sequences, the number of G.A mismatches, and the mismatch sequence order (5'-GA-3' or 5'-AG-3') affect the duplex stability. Changing A.T to G.C base pairs in sequences flanking the mismatches stabilizes the duplexes, but only to the extent observed with B-form DNA. The sequence 5'-pyrimidine-GA-purine-3', however, is considerably more stable than 5'-purine-GA-pyrimidine-3'. The most stable sequences with two pairs of adjacent G.A mismatches have thermodynamic parameters for duplex formation that are comparable to those for fully Watson-Crick base-paired duplexes. Similar sequences with single G.A pairs are much less stable than sequences with adjacent G.A mismatches. Reversing the mismatch order from 5'-GA-3' to 5'-AG-3' results in an oligomer that does not form a duplex. These results agree with predictions from the model derived from NMR and molecular mechanics and indicate that the sequence 5'-pyrimidine-GA-purine-3' forms a stable conformational unit that fits quite well into a B-form double helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peptide nucleic acid-DNA duplexes containing the universal base 3-nitropyrrole

A peptide nucleic acid (PNA) monomer containing the universal base 3-nitropyrrole was synthesized by coupling 1-carboxymethyl-3-nitropyrrole to ethyl N-[2-(tert-butoxycarbonylamino)ethyl]glycinate. The PNA sequence H-TGTACGTXACAACTA-NH2 (X = 3-nitropyrrole and C) and DNA sequence 5'-TGTACGTXACAACTA-3' were synthesized and thermal melting studies with the complementary DNA sequence 5'-TAGTTGTYACGTACA-3' (Y = A,C, G, T) compared. The T(m) data show that 3-nitropyrrole pairs indiscriminately with all four natural nucleobases as a constituent of either DNA or PNA. However, 3-nitropyrrole-containing PNA-DNA (average T(m) value = 51.1 degrees C) is significantly more thermally stable than 3-nitropyrrole-containing DNA-DNA (average T(m) value = 39.6 degrees C). From circular dichroism measurements, it is apparent that 3-nitropyrrole in the PNA strand causes a significant change in duplex structure.     

Effect of sequence context on O(6)-methylguanine repair and replication in vivo.

Understanding the origins of mutational hotspots is complicated by the intertwining of several variables. The selective formation, repair, and replication of a DNA lesion, such as O(6)-methylguanine (m(6)G), can, in principle, be influenced by the surrounding nucleotide environment. A nearest-neighbor analysis was used to address the contribution of sequence context on m(6)G repair by the Escherichia coli methyltransferases Ada or Ogt, and on DNA polymerase infidelity in vivo. Sixteen M13 viral genomes with m(6)G flanked by all permutations of G, A, T, and C were constructed and individually transformed into repair-deficient and repair-proficient isogenic cell strains. The 16 genomes were introduced in duplicate into 5 different cellular backgrounds for a total of 160 independent experiments, for which mutations were scored using a recently developed assay. The Ada methyltransferase demonstrated strong 5' and 3' sequence-specific repair of m(6)G in vivo. The Ada 5' preference decreased in the general order: GXN > CXN > TXN > AXN (X = m(6)G, N = any base), while the Ada 3' preference decreased in the order: NX(T/C) > NX(G/A), with mutation frequencies (MFs) ranging from 35% to 90%. The Ogt methyltransferase provided MFs ranging from 10% to 25%. As was demonstrated by Ada, the Ogt methyltransferase repaired m(6)G poorly in an AXN context. When both methyltransferases were removed, the MF was nearly 100% for all sequence contexts, consistent with the view that the replicative DNA polymerase places T opposite m(6)G during replication irrespective of the local sequence environment.     

Partial purification and cleavage specificity of a site-specific endonuclease, SciNI, isolated from Spiroplasma citri

A site-specific endonuclease, SciNI, has been partially purified from the plant pathogen Spiroplasma citri. The enzyme recognizes the sequence 5'-G-C-G-C-3' and cleaves between the first G and C. 3'-C-G-C-G-5' SciNI is an isoschizomer of HhaI, but generates DNA fragments with 5' rather than 3' single-stranded protrusions.     

A comparison of the different DNA binding specificities of the bZip proteins C/EBP and GCN4.

The bZip proteins GCN4 and C/EBP differ in their DNA binding specificities: GCN4 binds well to the pseudopalindromic AP1 site 5'-A4T3G2A1C0T1C2'A3'T4'-3' and to the palindromic ATF/CREB sequence 5'-A4T3G2A1-C0*G0'T1'C2'A3'T4'-3'; C/EBP preferentially recognizes the palindromic sequence 5'-A4T3T2G1C0*G0'C1'A2'-A3'T4'-3'. According to the X-ray structures of GCN4-DNA complexes, five residues of the basic region of GCN4 are involved in specific base contacts: asparagine -18, alanine -15, alanine -14, serine -11 and arginine -10 (numbered relative to the start point of the leucine zipper, which we define as +1). In the basic region of C/EBP position -14 is occupied by valine instead of alanine, the other four residues being identical. Here we analyse the role of valine -14 in C/EBP-DNA complex formation. Starting from a C/EBP-GCN4 chimeric bZip peptide which displays C/EBP specificity, we systematically mutated position -14 of its basic region and characterized the DNA binding specificities of the 20 possible different peptides by gel mobility shift assays with various target sites. We present evidence that valine -14 of C/EBP interacts more strongly with thymine 2 than with cytosine 1' of the C/EBP binding site, unlike the corresponding alanine -14 of GCN4, which exclusively contacts thymine 1' of the GCN4 binding sites.     

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.     

Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: alteration of DNA binding specificity through alteration of DNA kinking.

The catabolite activator protein (CAP) sharply bends DNA in the CAP-DNA complex, introducing a DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' ("primary kink"). CAP recognizes the base-pair immediately 5' to the primary-kink site, T:A(6), through an "indirect-readout" mechanism involving sequence effects on the energetics of primary-kink formation. CAP recognizes the base-pair immediately 3' to the primary-kink site, G:C(7), through a "direct-readout" mechanism involving formation of a hydrogen bond between Glu181 of CAP and G:C(7). Here, we report that substitution of the carboxylate side-chain of Glu181 of CAP by the one-methylene-group-shorter carboxylate side-chain of Asp changes DNA binding specificity at position 6 of the DNA half site, changing specificity for T:A(6) to specificity for C:G(6), and we report a crystallographic analysis defining the structural basis of the change in specificity. The Glu181-->Asp substitution eliminates the primary kink and thus eliminates indirect-readout-based specificity for T:A(6). The Glu181-->Asp substitution does not eliminate hydrogen-bond formation with G:C(7), and thus does not eliminate direct-readout-based specificity for G:C(7).     

Branch capture reactions: effect of recipient structure.

Branch capture reactions (BCR) contain two DNA species: (i) a recipient restriction fragment terminating in an overhang and (ii) a displacer-linker duplex terminating in a displacer tail complementary to the overhang as well as contiguous nucleotides within the recipient duplex. Branched complexes containing both species are captured by ligation of the linker to the recipient overhang. Specificity depends upon branch migration and is increased by substitution of bromodeoxycytidine for deoxycytidine in the displacer. BCR rates and specificities were determined for recipient overhangs that were (i) 5' and 3', (ii) 3 and 4 nucleotides long, and (iii) 0-100% G+C. Model systems permitted independent determination of G+C and branching effects on ligation rates and verification of rapid equilibrium between the branched complex and its component species. With all 4-base overhangs, recipient duplexes permitting extensive branch migration became saturated with displacer-linker duplexes. With increasing G+C, increasing ligation at competing sites led to decreased BCR specificity. BCR may be used to label a DNA fragment prior to electrophoresis, mark a fragment for affinity chromatography, or introduce a new overhang sequence compatible with a restriction endonuclease site in a cloning vector. A protocol was confirmed for mapping restriction sites in cloned DNA.     

DNA Sequence Effects on Single Base Deletions Arising during DNA Polymerization in Vitro by Escherichia Coli Klenow Fragment Polymerase

Most single base deletions detected after DNA polymerization in vitro directed by either Escherichia coli DNA polymerase I or its Klenow fragment are opposite Pu in the template. The most frequent study, were previously found to be associated with the consensus template context 5'-PyTPu-3'. In this study, the predictive power of the consensus sequence on single base deletion frequencies was directly tested by parallel comparison of mutations arising in four related DNAs differing by a single base. G, a deletion hotspot within the template context 5'-TTGA-3', was substituted by each of the 3 other bases. Previous studies had shown that deletions opposite the G were frequent but that deletions opposite its neighboring A were never detected. Based on the predictions of the consensus, the substitution of T for G should produce frequent deletions opposite the neighboring A due to its new 5'-TTTA-3' template context. This prediction was fulfilled; no deletions of this A were detected in the other templates. The consensus further predicted that deletions opposite template C would be lower than those opposite either A or G at the same site and this prediction was also fulfilled. The C substitution also produced a new hotspot for 1 bp deletions 14 bp away. The new hotspot depends on quasi-palindromic misalignment of the newly synthesized DNA strand during polymerization; accurate, but ectopically templated synthesis is responsible for this mutagenesis. Mutations templated by quasi-palindromic misalignments have previously been recognized when they produced complex sequence changes; here we show that this mechanism can produce frequent single base deletions. The unique stimulation of misalignment mutagenesis by the C substitution in the template is consistent with the singular ability of C at that site to contribute to extended complementary pairing during the DNA misalignment that precedes mutagenesis.     

General nearest neighbor preferences in G/C oligomers interrupted by A/T: correlation with DNA structure

The frequencies of occurrence of the 5' and 3' nearest neighbor doublets of oligonucleotides containing (G/C) and (A/T) blocks show strong trends. Specifically, the following trends are observed. Given a (G/C)n (A/T)m oligomer (where G/C)n indicates a sequence of length n composed solely of Gs and/or Cs and (A/T)m is a sequence of length m composed solely of As and/or Ts, and n = 3,2,1; m = 1,2,3) and a (G/mC)2 doublet, (G/C)n (A/T)m (G/C)2 greater than (G/C)n + 2 (A/T)m. That is the (G/C)2 doublet is preferentially located 3' of the oligomer, enclosing the (A/T)m stretch. The trends are strongest for n = 3, m = 1 and gradually weaken as the size of the (mG/C)n block decreases (with a concomitant increase of (A/T)m). (A/T)2 nearest neighbor flank preferentially encloses the (G/C)n block (to produce (A/T)2 (G/C)n (A/T)m). The (A/T)2 flank trends are weaker than the (G/C)2 flank ones. The (A/T)2 flank trends also decrease in strength as the size of the (G/C)n block decreases. The statistical significance of these trends in eukaryotes is very high. A possible correlation with DNA structural parameters, in particular groove geometry, is discussed.     

Sequence requirements for mammalian topoisomerase II mediated DNA cleavage stimulated by an ellipticine derivative.

Various antitumor drugs stabilize DNA topoisomerase II-DNA transient covalent complexes. The complexes distribution along pBR322 DNA was shown previously to depend upon the nature of the drug (Tewey et al. (1984) Science 226, 466-468). The position in pBR322 of DNA cleavage by calf DNA topoisomerase II for 115 such sites stabilized by an ellipticine derivative and the relative frequency of cleavage at most of these sites were determined. The nucleotide sequence surrounding the 25 strongest sites was analyzed and the following ellipticine specific consensus sequence was deduced: 5'-ANCNT(A/G)T.NN(G/C)N(A/G)-3' where cleavage occurs at the indicated mark. A thymine is always present at the 3' end of at least one strand of the strong cleavage sites, and the dinucleotide AT or GT at the 3' end of the break plays a major role in the complex stabilisation. The predictive value of cleavage of the consensus was tested for two regions of SV40 DNA and cleavage was indeed detected at the majority of the sites matching the consensus. Some complexes stabilized by ellipticine are resistant to salt dissociation and this property seems to be correlated with the presence of symmetrical sequences in the cleavage site with a center of symmetry staggered relatively to the center of symmetry of cleavage.     

Some restriction endonucleases tolerate single mismatches of the pyrimidine.purine type.

DNAs from phage mutants M13mp18 and M13mp18/MP-1 were used to construct two closed circular heteroduplexes. One of them carried the sequence 5'-CCTGGG-3' 3'-GGGCCC-5' with a T.G mismatch at the position 6248. The other carried the sequence 5'-CCCGGG-3' 3'-GGACCC-5' with a C.A mismatch at the same position. Heteroduplexes were exposed to 7 restriction endonucleases having recognition sites within the sequence 5'-CCCGGG-3' 3'-GGGCCC-5' and to 1 restriction endonuclease having a recognition site within the sequence 5'-CCTGGG-3' 3'-GGACCC-5'. All tested enzymes cleaved at least one mismatch-containing sequence although with reduced efficiency. Smal and Xmal tolerated both mismatch-containing sequences. Aval, Hpall, Mspl, Ncil and Nsplll were able to tolerate only the T.G containing sequence, while BstNl was able to tolerate only the C.A containing sequence. It is inferred that the tolerance displayed by Smal and Xmal depends on the presence of either the original purines or the original pyrimidines in mismatches of both the T.G and C.A type and that all other tested enzymes require the presence of the original purines in mismaches of both types.