Cleavage of single strand oligonucleotides and bacteriophage phi X174 DNA by Msp I endonuclease

Type II restriction endonucleases cleave duplex DNA at nucleotide sequences displaying 2-fold symmetry. Our data show that Msp I cleaves single strand oligonucleotides, d(G-A-A-C-C-G-G-A-G-A) and d(T-C-T-C-C-G-G-T-T) at 4 degrees, 25 degrees, and 37 degrees C reaction temperatures. The rate of cleavage of d(G-A-A-C-C-G-G-A-G-A) is several-fold faster than that of d(T-C-T-C-C-G-G-T-T). Single strand phi X174 DNA is also, cleaved by Msp I endonuclease giving well defined fragments. 5'-Nucleotide analysis of the fragments generated from single strand and replicating form DNA suggest that cleavage occurs at the recognition sequence d(C-C-G-G). The data show that Msp I endonuclease cleaves single strand oligonucleotides and prefers a recognition sequence surrounded by purine nucleotides. A general model for endonuclease cleavage of single strand and duplex DNA is presented.     

The influence of the exocyclic amino group characteristic of GC base pairs on molecular recognition of specific nucleotide sequences in DNA by Berenil and DAPI

The expedient of preparing homologous DNA samples substituted with inosine for guanosine residues, 2,6-diaminopurine (DAP) for adenine residues, or both, has been used to investigate the role of the purine 2-amino group in determining the preferred binding sites for the drugs berenil [1,3-bis(4-phenylamidinium) triazene] and DAPI (4′,6-diamidino-2-phenyl indole) on DNA. The selectivity of these two minor groove binders for AT-rich sequences is seen to be radically altered in the substituted DNA molecules. Neither berenil nor DAPI bind to DAP-substituted DNA where all purine residues bear a 2-amino group. By contrast, they bind to AT-rich, IC-rich and even mixed sequences of the inosine DNA where all purine residues lack the 2-amino group. With the inosine and DAP double substituted DNA, both berenil and DAPI bind preferentially to IC-rich clusters instead of their canonical tracts endowed with an extra 2-amino group through substitution with DAP. These results establish that the location of the purine 2-amino group represents a critical determinant for recognition of DNA nucleotide sequences by the two drugs. © 1997 John Wiley & Sons, Ltd.     

DNA-probes for the highly sensitive identification of single nucleotide polymorphism using single-molecule spectroscopy

This article presents a new, highly sensitive method for the identification of single nucleotide polymorphisms (SNPs) in homogeneous solutions using fluorescently labeled hairpin-structured oligonucleotides (smart probes) and fluorescence single-molecule spectroscopy. While the hairpin probe is closed, fluorescence intensity is quenched due to close contact between the chromophore and several guanosine residues. Upon hybridization to the respective target SNP sequence, contact is lost and the fluorescence intensity increases significantly. High specificity is achieved by blocking sequences containing mismatch with unlabeled oligonucleotides. Time-resolved single-molecule fluorescence spectroscopy enables the detection of individual smart probes passing a small detection volume. This method leads to a subnanomolar sensitivity for this single nucleotide specific DNA assay technique.     

Parallel self-associated structures formed by T,C-rich sequences at acidic pH

Oligonucleotides of nonregular heteropyrimidine sequences incorporating or not incorporating purine residues 5'-d(ACTCCCTTCTCCTCTCTA), 5'-d(ACTCCCTGGTCCTCTCTA), 5'-d(TCTCTCCTGGTCCCTCC), and 5'-d(TCTCTCCTCTTCCCTCC) can form self-associated parallel-stranded (ps) structures at pH 4-5.5. The ps structures were identified by studying at neutral and acidic pH UV melting transitions, FTIR spectra, and fluorescence of pyrene-labeled oligonucleotides as well as by chemical joining of 5'-phosphorylated oligonucleotides. A gel electrophoresis run for oligonucleotides 5'-d(TCTCTCCTCTTCCCTCC) and 5'-d(ACTCCCTTCTCCTCTCTA) has shown the formation of homoduplexes at low DNA strand concentrations. Ps structures are held by C-C(+) base pairs and have N- and S-types of sugar puckering as detected by FTIR spectroscopy in the millimolar concentration range. Guanine inserts as well as thymine and purine inserts into an oligomeric cytosine sequence make the formation of the tetraplex i-motif unfavorable. MvaI restriction endonuclease, which recognizes the CCT/AGG sequence in DNA, does not cleave parallel pseudosubstrates.     

On the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of [Me-3H]methionine, practically all the radioactivity incorporated into DNA is found to exist in 5-methylcytosine and N6-methyladenine. The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of 5-methylcytosine in R-m5 C-R and R-m5 C-T-R oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring 5-methylcytosine to be identified and shows that 5-methylcytosine localizes in G-m5 C-A and G-m5 C-Tr fragments. B. brevis S DNA methylase modifying cytosine residues recognizes the GCA/TGC degenerate nucleotide sequence which is a part of the following complementary structure with a two-fold rotational axis of symmetry: (5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5') (Methylated cytosine residues are askerisked). Cytosine-modifying DNA methylase activity is isolated from B. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence DNA in bacterial cells can be undermethylated. This enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA. DNA methylases of different variants of B. brevis (R, S, P+, P-)) methylate cytosine residues in the same nucleotide sequences. It means that specificity or methylation of DNA cytosine residues in the cells of different variants of B. brevis is the same.     

Site-specific DNA cleavage by antisense oligonucleotides covalently linked to phenazine di-N-oxide.

Site-specific degradation of DNA was achieved by the use of DNA oligonucleotides covalently tethered to phenazine 5,10-di-N-oxide. When annealed to a complementary DNA target strand, the antisense oligonucleotide effected alkylation of guanosine residues in proximity to the phenazine di-N-oxide prosthetic group. Admixture of dithiothreitol to the formed duplex resulted in reductive activation of the phenazine di-N-oxide moiety with concomitant generation of diffusible oxygen radicals; the latter effected strand scission of the target DNA oligonucleotide. Several parameters of DNA degradation were studied, including the effect on DNA degradation of chain length in the tether connecting the oligonucleotides and prosthetic group, the relative efficiencies of DNA cleavage when the prosthetic group was in the middle or at the end of the antisense oligonucleotide, and the effect of O2 on DNA degradation. Also studied was the actual chemistry of DNA oligonucleotide degradation and the ability of individual diastereomers of the modified oligonucleotides to mediate degradation of the target DNA.     

Rational design of switched triple helix-forming oligonucleotides: Extension of sequences for triple helix formation

A rational design by means of molecular mechanics has been carried out in an effort to extend the range of double-helical DNA sequences that could be recognized by triple helix-forming oligonucleotides. The DNA target is composed of alternating, adjacent fragments of oligopurine·oligopyrimidine sequences, instead of a long stretch of polypurine·polypyrimidine sequence used for canonical triple helix formation. Based on the combination of different triple helix motifs in eitherHoogsteen orreverse Hoogsteen configuration, mini-triple helices can be formed at each oligopurine·oligopyrimidine part of the target sequence with either parallel or antiparallel orientation with respect to the purine strand. As the adjacent purine target sequences are located in the complementary strands, the third strand oligonucleotides can be joined together through a natural phosphodiester backbone at the junctions in either a 5-3 or a 3-5 polarity. There are six distinct junction steps. Molecular modeling was aimed at optimizing the cooperative binding of the so-called switched triple helix-forming oligonucleotides by choosing appropriate nucleotide(s) at the junction between two adjacent minitriple helices. A comprehensiveswitch code describing the rules for forming switched triple helices has been established. Its practical applications in extending DNA recognition by this new generation of tailor-made triple helix-forming oligonucleotides are discussed.     

Specificity of methylation of cytosine risidues in DNA of Bacillus brevis var. G-B]

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of (methyl-3H)-methionine practically the total radioactivity included into DNA is found to exist in 5-methylcytosine (MC) and 6N-methyladenine (MA). The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of MC in Pur-MC-Pur and Pur-MC-T-Pur oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring with MC to be revealed and shows that MC localizes in G-MC-A and G-MC-T-Pu fragments. Bac. brevis S DNA-methylase modifying cytosine residues recognizes the GCAT GC degenerative nucleotide sequence which is a part of the following complementary structure with rotational symmetry: (5') ... N'--G--MC--T--G--C--N ... (3') (3') ... N--C--G--A--MC--G--N' ... (5') Cytosine modifying DNA-methylase activity is isolated from Bac. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence, DNA in bacterial cells can be partially undermethylated. This enzyme methylates cytosine residues in native and deneaturated DNA in the same nucleotide sequences. As compared to the native DNA, the denaturated DNA is indicative of a decrease in the level of methylation of adenine, rather than cytosine residues. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA (calf thymus, Pseudomonas aeruginosa etc.). DNA-methylases of different variants of Bac. brevis (R, S, P+, P-) methylate cytosine residues in the same nucleotide sequences. It means that specificity of methylation of DNA cytosine residues in the cells of different variants of Bac. brevis is the same.     

Nucleotide sequence and properties of the cohesive DNA termini from bacteriophage HP1c1 of Haemophilus influenzae Rd

The termini of the mature DNA of phage HP1c1 of Haemophilus influenzae Rd have been characterized by DNA ligation, nucleotide sequencing, and deoxynucleotide incorporation experiments. A hybrid plasmid containing the joined phage termini (the cos site) inserted into pBR322 has been constructed. The phage DNA has cohesive termini composed of complementary 5' single-stranded extensions which are seven residues long. The left cohesive terminal extension consists only of pyrimidines and the right only of purines. When the ends of the phage are joined, the terminal sequences constitute the central 7 bp of an 11 bp sequence containing only purines on one strand and pyrimidines on the other strand. This oligopyrimidine/oligopurine sequence does not possess rotational symmetry. A 10-bp sequence and its inverted repeat are located approx. 20 bp to the left and right of the fused ends.     

A method for introducing random single point deletions in specific DNA target sequences using oligonucleotides.

We describe a method for the generation of random point deletions in any target DNA sequence using synthetic mixed oligonucleotides. A mixed pool of oligonucleotides, which contain single nucleotide deletions randomly distributed throughout the full length, was generated by a modification of the synthesis cycle of an automated DNA synthesiser that allowed the inefficient incorporation of nucleotide monomers during each cycle of synthesis. A family of oligonucleotides was used to prime in vitro synthesis of the complementary strand of a cloned DNA fragment in an M13 vector which had previously been passaged through a dut-, ung- Escherichia coli host. Strong selection for progeny from the newly synthesised strand is provided by transforming the heteroduplex into a dut+, ung+ host. This procedure introduced point deletions at 10-25% efficiency. It has been used to introduce point deletions into operator sequences which bind the yeast regulatory proteins encoded by MATa1 and MAT alpha 2.     

Temporal patterns of nucleotide misincorporations and DNA fragmentation in ancient DNA

DNA that survives in museum specimens, bones and other tissues recovered by archaeologists is invariably fragmented and chemically modified. The extent to which such modifications accumulate over time is largely unknown but could potentially be used to differentiate between endogenous old DNA and present-day DNA contaminating specimens and experiments. Here we examine mitochondrial DNA sequences from tissue remains that vary in age between 18 and 60,000 years with respect to three molecular features: fragment length, base composition at strand breaks, and apparent C to T substitutions. We find that fragment length does not decrease consistently over time and that strand breaks occur preferentially before purine residues by what may be at least two different molecular mechanisms that are not yet understood. In contrast, the frequency of apparent C to T substitutions towards the 5'-ends of molecules tends to increase over time. These nucleotide misincorporations are thus a useful tool to distinguish recent from ancient DNA sources in specimens that have not been subjected to unusual or harsh treatments.     

Different conformational families of pyrimidine.purine.pyrimidine triple helices depending on backbone composition.

Different helical conformations of DNA (D), RNA (R), and DNA.RNA (DR) hybrid double and triple helices have been detected using affinity cleavage analysis. Synthetic methods were developed to attach EDTA.Fe to a single nucleotide on RNA as well as DNA oligonucleotides. Cleavage patterns generated by a localized diffusible oxidant in the major groove on the pyrimidine strand of four purine.pyrimidine double helices consisting of all DNA, all RNA, and the corresponding hybrids reveal that the relative cleavage intensity shifts to the 5' end of the purine strand increasingly in the order: DD < DR < RD < RR. These results are consistent with models derived from structural studies. In six pyrimidine.purine.pyrimidine triple helices, the altered cleavage patterns of the Watson-Crick pyrimidine strands reveal at least two conformational families: (i) D + DD, R + DD, D + DR, and R + DR and (ii) R + RD and R + RR.     

Both substrate and target oligonucleotide sequences affect in vitro integration mediated by human immunodeficiency virus type 1 integrase protein produced in Saccharomyces cerevisiae.

Integration of retroviral DNA into the host cell genome requires the interaction of retroviral integrase (IN) protein with the outer ends of both viral long terminal repeats (LTRs) to remove two nucleotides from the 3' ends (3' processing) and to join the 3' ends to newly created 5' ends in target DNA (strand transfer). We have purified the IN protein of human immunodeficiency virus type 1 (HIV-1) after production in Saccharomyces cerevisiae and found it to have many of the properties described for retroviral IN proteins. The protein performs both 3' processing and strand transfer reactions by using HIV-1 or HIV-2 attachment (att) site oligonucleotides. A highly conserved CA dinucleotide adjacent to the 3' processing site of HIV-1 is important for both the 3' processing and strand transfer reactions; however, it is not sufficient for full IN activity, since alteration of nucleotide sequences internal to the HIV-1 U5 CA also impairs IN function, and Moloney murine leukemia virus att site oligonucleotides are poor substrates for HIV-1 IN. When HIV-1 att sequences are positioned internally in an LTR-LTR circle junction substrate, HIV-1 IN fails to cleave the substrate preferentially at positions coinciding with correct 3' processing, implying a requirement for positioning att sites near DNA ends. The 2 bp normally located beyond the 3' CA in linear DNA are not essential for in vitro integration, since mutant oligonucleotides with single-stranded 3' or 5' extensions or with no residues beyond the CA dinucleotide are efficiently used. Selection of target sites is nonrandom when att site oligonucleotides are joined to each other in vitro. We modified an in vitro assay to distinguish oligonucleotides serving as the substrate for 3' processing and as the target for strand transfer. The modified assay demonstrates that nonrandom usage of target sites is dependent on the target oligonucleotide sequence and independent of the oligonucleotide used as the substrate for 3' processing.     

Steady‐state and time‐resolved fluorescence of tetramethylrhodamine attached to DNA: correlation with DNA sequences

Time‐resolved fluorescence as well as steady‐state absorption and fluorescence were detected in order to study the interactions between tetramethylrhodamine (TAMRA) and DNA when TAMRA was covalently labeled on single‐ and double‐stranded oligonucleotides. Fluorescence intensity quenching and lifetime changes were characterized and correlated with different DNA sequences. The results demonstrated that the photoinduced electron transfer interaction between guanosine residues and TAMRA introduced a short lifetime fluorescence component when guanosine residues were at the TAMRA‐attached terminal of the DNA sequences. The discrepancy of two‐state and three‐state models in previous studies was due to the DNA sequence selection and sensitivity of techniques used to detect the short lifetime component. The results will help the design of fluorescence‐based experiments related to a dye labeled probe. Copyright © 2012 John Wiley & Sons, Ltd.     

An oligopurine sequence bias occurs in eukaryotic viruses.

Twenty four DNA and RNA viral nucleotide sequences, comprising over 346 kilobases, have been analyzed for the occurrence of strings of contiguous purine or pyrimidine residues. On average strings greater than or equal to 10 contiguous purines or pyrimidines are found three and a half times more frequently than would be expected for a random distribution of bases. Detailed analysis of the 172 kilobase Epstein-Barr viral sequence shows that the bias in favor of contiguous purine residues increases with the length of the purine string. These findings are similar to those seen for genomic DNA from higher eukaryotes. In contrast no overrepresentation of oligopurine or oligopyrimidine strings is observed in 52 kilobases from eight bacteriophage and E. coli DNA sequences.     

Preferential recognition of I.T base-pairs in the initiation of excision-repair by hypoxanthine-DNA glycosylase.

Double-stranded synthetic oligonucleotides with a centrally located dIMP residue in a 5'-32P-labeled strand were employed as substrates for hypoxanthine-DNA glycosylase. The enzyme activity was monitored by the generation of a piperidine-sensitive site in the labeled oligonucleotide. The enzyme was purified approximately 5000-fold from calf thymus. The purified enzyme removed efficiently a hypoxanthine base residue from an I.T base pair, but 15-20 times more slowly from an I.C base pair. Similar results were obtained with oligonucleotides in which the deoxyinosine residue was placed in different surrounding nucleotide sequences. The enzyme had no detectable activity on mismatched G.T, A.G or A.C base pairs. The data indicate that hypoxanthine-DNA glycosylase participates in the repair of deaminated adenine residues in DNA.     

Determination of a nucleotide sequence in bacteriophage f1 DNA by primed synthesis with DNA polymerase

A method is described for the determination of nucleotide sequences in DNA by using specific oligonucleotides as primers for copying specific regions by DNA polymerase. The method was applied to bacteriophage f1 DNA using the synthetic octanucleotide A-C-C-A-T-C-C-A as primer and a sequence (sequence A) of 81 nueleotides was determined. Synthesis was carried out in the presence of manganese and with one of the deoxyribotriphosphates (dCTP or dGTP) replaced by the corresponding ribotriphosphate so that mixed oligonucleotides were found which could be specifically split at the ribonucleotide residues by the appropriate ribonuclease or by alkali. The relative order of the digestion products was determined by fractionating the undigested oligonucleotides according to size on a two-dimensional system and digesting the isolated products. In the presence of rGTP the octanucleotide appeared to prime at a second site giving rise to a second sequence (B) besides sequence A. The complementary sequence to sequence A, which corresponds to the plus strand of f1 DNA and to the messenger RNA, contains five nonsense codons, four of which are in the same phase, and two possible initiation codons. It also contains a repetitive sequence which suggests its evolutionary origin by duplication.     

Relative stabilities of triple helices composed of combinations of DNA, RNA and 2'-O-methyl-RNA backbones: chimeric circular oligonucleotides as probes.

Described is a systematic study of the effects of varied backbone structure on the stabilities of pyr.pur.pyr triple helices. The effects were measured using six circular 34 base oligonucleotides containing DNA (D), RNA (R) and/or 2'-O-methyl-RNA (M) residues designed to bind a complementary single-stranded purine target strand by triple helix formation. Eighteen different backbone combinations were studied at pH 5.5 and 7.0 by optical melting experiments and the results compared with the stabilities of the corresponding Watson-Crick duplexes. When the target purine strand is DNA, all circles form pH-dependent triple helical complexes which are considerably stronger than the duplexes alone. When RNA is the target, five of the nine complexes studied are of the pH-dependent triplex type and the other four complexes are not significantly stronger than the corresponding duplexes. The results are useful in the design of the highest affinity ligands for single- and double-stranded DNAs and RNAs and also point out novel ways to engender DNA- or RNA-selective binding.