Two sequence-specific endonucleases from Moraxella bovis

Two new sequence-specific endodeoxyribonucleases have been partially purified from Moraxella bovis. These restriction-like enzymes, MboI and MboII, each cleave bacteriophage lambda DNA and adenovirus-2 DNA at more than 50 sites. MboI recognizes the sequence 5′ ↓ G-A-T-C 3′ 3′ C-T-A-G ↑ 5′ and cleaves at the sites indicated by the arrows. A specific endonuclease, MosI, has also been purified from Moraxella osloenis and recognizes the same sequence as MboI.     

A specific endonuclease from Brevibacterium albidum

A new restriction-like endonuclease, BalI, has been partially purified from Brevibacterium albidum. This enzyme cleaves bacteriophage λ DNA at least 18 times and adenovirus-2 DNA at least 16 times, but does not cleave simian virus 40 DNA. All sites cleaved by BalI are also cut by the specific endonuclease HaeIII from Haemophilus aegyptius. The recognition sequence of BalI is 5′-T-G-G ↓ C-C-A-3′ 3′-A-C-C ↓ G-G-T-5′ and the cleavage site is indicated by the arrows.     

The specific non-symmetrical sequence recognized by restriction endonuclease MboII

The restriction endonuclease MboII, isolated from Moraxella bovis (ATCC 10900), cleaves bacteriophage φX174am3 replicative form I DNA into ten fragments. The physical map of these fragments has been aligned with the sequence of φX174 DNA. There is no sequence with 2-fold rotational symmetry common to the region of all ten cleavage sites. However, the non-symmetrical sequence 5′-G-A-A-G-A-3′ 3′-C-T-T-C-T-5′ occurs near to each cleavage site. Precise mapping of the cleavages in both DNA strands at several sites places the cuts eight nucleotides to the right of the upper sequence and seven nucleotides to the right of the lower sequence.     

A specific endonuclease from Arthrobacter luteus.

A new restriction-like endonuclease, AluI, has been partially purified from Arthrobacter luteus. This enzyme cleaves bacteriophage λ DNA, adenovirus-2 DNA and simian virus 40 DNA at many sites including all sites cleaved by the endonuclease HindIII from Haemophilus influenzae serotype d. Radioactive oligonucleotides in pancreatic DNAase digests of (5′-³²P)-labelled fragments of phage λ DNA released by the action of AluI had the 5′ terminal sequence pC-T-N-. The enzyme recognises the tetranucleotide sequence

and cleaves it at the position marked by the arrows.     

A sequence specific endonuclease from Micrococcus radiodurans

A new sequence specific endonuclease, MraI has been purified from Micrococcus radiodurans. This enzyme cleaves bacteriophage λ DNA at three sites, adenovirus type 2 DNA at more than 12 sites and has a unique site on ΦX174 DNA. It has no sites on SV40, PM2 and pBR322 DNA. The three sites on phage λ DNA are different from those cleaved by SmaI, XmaI and XorII. The sites of cleavage are located at 0.424, 0.447 and 0.834 fractional lengths on the physical map of λ DNA. MraI is shown to be an isoschizomer of SacII and SstII recognizing the palindromic nucleotide sequence ′5-CCGC↓GG-3′. The enzyme shows an absolute requirement of Mg²⁺, but is active in the absence of added 2-mercaptoethanol. The enzyme shows activity at a broad range of temperature and pH with an optimum at 45°C and pH 7.0. MraI represents the first restriction enzyme from a bacterium whose DNA lacks modified methylated bases.     

DNA recognition and cleavage by the EcoP15 restriction endonuclease.

EcoP15 is a restriction-modification enzyme coded by the P15 plasmid of Escherichia coli. We have determined the sites recognized by this enzyme on pBR322 and simian virus 40 DNA. The enzyme recognizes the sequence:

In restriction, the enzyme cleaves the DNA 25 to 26 base-pairs 3′ to this sequence to leave single-stranded 5′ protrusions two bases long.     

The DNA sequence recognised by the HinfIII restriction endonuclease

HinfIII is a type III restriction enzyme (Kauc &; Piekarowicz, 1978) isolated from Haemophilus influenzae Rf. Like other type III restriction endonucleases, the enzyme also catalyses the modification of susceptible DNA. It requires ATP for DNA cleavage and S-adenosyl methionine for DNA methylation. We have determined the DNA sequence recognised by HinfIII to be:

$\text{5′-C-G-A-A-T-3′}\text{·····3′-G-C-T-T-A-5′}$

In restriction, the enzyme cleaves the DNA about 25 base-pairs to the right of this sequence. In the modification reaction only one of the strands is methylated, that containing the 5′-C-G-A-A-T-3′ sequence.     

Processing of the abasic sites clustered with the benzo[a]pyrene adducts by the base excision repair enzymes

The major enzyme in eukaryotic cells that catalyzes the cleavage of apurinic/apyrimidinic (AP or abasic) sites is AP endonuclease 1 (APE1) that cleaves the phosphodiester bond on the 5′-side of AP sites. We found that the efficiency of AP site cleavage by APE1 was affected by the benzo[a]pyrenyl-DNA adduct (BPDE-dG) in the opposite strand. AP sites directly opposite of the modified dG or shifted toward the 5′ direction were hydrolyzed by APE1 with an efficiency moderately lower than the AP site in the control DNA duplex, whereas AP sites shifted toward the 3′ direction were hydrolyzed significantly less efficiently. For all DNA structures except DNA with the AP site shifted by 3 nucleotides in the 3′ direction (AP₊₃-BP-DNA), hydrolysis was more efficient in the case of (+)-trans-BPDE-dG. Using molecular dynamic simulation, we have shown that in the complex of APE1 with the AP₊₃-BP-DNA, the BP residue is located within the DNA bend induced by APE1 and contacts the amino acids in the enzyme catalytic center and the catalytic metal ion. The geometry of the APE1 active site is perturbed more significantly by the trans-isomer of BPDE-dG that intercalates into the APE1-DNA complex near the cleaved phosphodiester bond. The ability of DNA polymerases β (Polβ), λ and ι to catalyze gap-filling synthesis in cooperation with APE1 was also analyzed. Polβ was shown to inhibit the 3′ → 5′ exonuclease activity of APE1 when both enzymes were added simultaneously and to insert the correct nucleotide into the gap arising after AP site hydrolysis. Therefore, further evidence for the functional cooperation of APE1 and Polβ in base excision repair was obtained.     

The relaxation of specificity of BanI restriction endonuclease from Bacillus aneurinolyticus IAM 1077

The restriction endonuclease BanI from Bacillus aneurinolyticus IAM 1077, which recognizes 5′-GGPyPuCC-3′ and cleaves between G and G within this sequence, has decreased substrate specificity at high nuclease concentrations. The relaxation of its specificity was enhanced during modified reactions: digestion of pBR322 DNA or lambda DNA in the presence of high glycerol and dimethyl-sulfoxide (DMSO) produced additional fragments in addition to the inherent fragments. Therefore, it is required to check the reaction conditions carefully for generation of inherent fragments.     

Characterization of DNA restriction and modification activities in Neisseria species

Type II restriction endonuclease activities detected in various Neisseria species were characterized for sequence specificity and precise site of cleavage. NsiCI isolated from N. sicca C351 cleaves the sequence 5′-GAT↓ATC-3′ (EcoRV isoschizomer); NmeCI from N. meningitidis C114 and NphI from N. pharyngis C245 cleave 5′-N↓GATCN-3′ (MboI isoschizomers); NgoPII and NgoPIII from N. gonorrhoeae P9-2 cleave at 5′-CC↓GCGG-3′ (SacII isoschizomer) and 5′-GG↓CC-3′ (HaeIII isoschizomer), respectively. Chromosomal DNA isolated from these strains and two other N. meningitidis strains (which lacked detectable endonuclease activities), was found to be refractive to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases.     

Nucleotide recognition seouence at the cleavage site of Haemophilus aegyptius II (Hae II) restriction endonuclease

We have determined the nucleotide sequence recognized by the restriction endonuclease Hae II from Haemophilus aegyptius which cleaves the simian virus 40 (SV40) DNA at a single specific site. By using terminal radioactive labeling of the cleavage site at both the 5′ and 3′-ends we have deduced the recognition sequence,

with elements of a two-fold rotational symmetry. The endonuclease produces staggered ends with protruding 3′-terminated single-strands, four nucleotides in length. In plasmid RSF 2124 DNA, which contains multiple Hae II cleavage sites, it was observed that the 5th nucleotide from the 3′ terminus is either a pdA or a pdG, indicating alternating recognition sequences.     

Characterization of a site-specific restriction endonuclease SphI from Streptomyces phaeochromogenes

A new type-II restriction endonuclease SphI, has been partially purified from Streptomyces phaeochromogenes. SphI recognizes the hexanucleotide sequence 5′-GCATG↓C and cleaves it at the position marked by the arrow. This nucleotide sequence is present twice in SV40 DNA, four times in λ DNA and only once in the cloning vehicles pBR322, pBR325, pBR327 and pBR328.     

Preliminary x-ray diffraction studies on lobster ATP: L-arginine phosphotransferase.

An endonuelease R.HindIII, prepared from Hemophilus influenzae strain Rd, degrades foreign DNA, but not homologous DNA. Phage T7 DNA is also resistant to the enzyme. Fragments of phage λ DNA produced by treatment with R.HindIII have been labelled at their 5′ termini and analysis of the radioactive nucleotides in pancreatic DNAase digests of these fragments revealed a single 5′ terminal sequence. From this and other data we conclude that the enzyme recognizes and cleaves DNA at the following nucleotide sequence,

giving termini bearing short cohesive ends.     

A second specific endonuclease from Haemophilus aegyptius.

A second restriction-like endonuclease has been partially purified from Haemophilus aegyptius. This enzyme cleaves bacteriophage λ DNA and adenovirus 2 DNA at many sites, but cleaves simian virus 40 DNA at only one site.     

The influence of linker chain length on the sequence specificity of DNA damage by n-bromoalkylphenanthridinium bromides in plasmid DNA and in intact human cells

The sequence specificity of DNA damage of n-bromoalkylphenanthridinium bromides, with linker chain lengths (n) of 4,6,8 and 10 methylene groups, was investigated in the plasmid pUC8 and in intact human cells. A linear amplification assay was used to elucidate the DNA sequence specificity of the alkylating agents. In this assay Taq DNA polymerase extends from an oligonucleotide primer up to the damage site and the products run on a DNA sequencing gel to reveal the precise sites of DNA damage. For both the plasmid and cellular experiments, the compound that caused the most damage to DNA was the n = 6 compound, followed by (in decreasing order) the n = 4, n = 8, and n = 10 compounds. There were significant differences in the sequence specificity of DNA damage between n-bromoalkylphenanthridinium bromides of different linker chain length: (1) the main sites of damage were at guanines for the n = 4,6 and 8 compounds but at guanines and adenines for the n = 10 compound; (2) a consensus sequence of 5′-c(a/t)Ggg-3′ was obtained for the n = 4,6 and 8 compounds but 5′-c(a/c)(G/A)(g/a)-3′ for the n = 10 compound; (3) runs of consecutive Gs were the major site of damage for the n = 4,6 and 8 compounds, but consecutive Gs or consecutive As for the n = 10 compound; (4) for damage at single isolated guanines, the most damaged sequences were at 5′-Ga-3′ for the n = 4 compound but at 5′-Gt-3′ for the n = 6,8 and 10 compounds. The tandemly repeated alpha RI DNA sequence was the DNA target in intact human K562 cells. In intact human cells, the compounds produced damage with similar DNA sequence selectivity to that found in plasmid DNA. The n = 4 and 6 compounds possess marginal anti-tumour activity and these compounds produced the most damage in intact human cells. The n = 8 and 10 compounds do not demonstrate significant anti-tumour activity and these compounds resulted in the least damage in cells.     

DNA sequence analysis of host range mutants of the promiscuous IncP-1 plasmids R18 and R68 with Tn7 insertions in oriV

Transposon Tn7 insertions in the origin of vegetative replication (oriV) result in host range mutants of the promiscuous IncP-1 plasmids R18 and R68 which affect plasmid replication in Escherichia coli but not in Pseudomonas aeruginosa. The sites of these insertions have been analyzed by DNA sequence analysis. In two mutants, the insertions generated direct duplications of 5′GTATT3′ at the target site which included the first base at the 5′ end of the fourth 17-bp direct repeat in oriV. In a third mutant the duplication of 5′GACAC3′ also involved the same direct repeat also at the 5′ end but contiguous with the previous duplication. DNA sequence analysis of another Tn7-induced host range mutant of R18, characterized by reduced conjugational transmissibility into P. stutzeri while retaining normal transmissibility within P. aeruginosa, showed that the insertion generated a 474-bp deletion which brought the insertion 20 bp 5′ to the 17-bp direct repeat between oriV and the oxytetracycline hydrochloride-resistant gene. The analysis of the DNA sequence data at the site of the Tn7 insertions shows that particular segments of the DNA sequence in oriV are differentially required for the replication of these plasmids in different bacterial hosts and thus of importance to the promiscuity of these plasmids.     

Expression,purification and biochemical characterization of Methanocaldococcus jannaschii DNA ligase

We describe the biochemical characterization of Methanocaldococcus jannaschii (M. jannaschii) DNA ligase and its potential application in single nucleotide polymorphism (SNP) genotyping. The recombinant M. jannaschii DNA ligase is an ATP-dependent ligase. The ligase activity was dependent on metal ions of Mg²⁺ and Mn²⁺. The optimal concentrations of ATP cofactor and Mg²⁺ ion were 0.01–2 and 10 mM, respectively. The optimal pH value for DNA ligation was 8.5. High concentrations of NaCl inhibited DNA ligation. The effects of mismatches on joining short oligonucleotides by M. jannaschii DNA ligase were fully characterized. The mismatches at the first position 5′ to the nick inhibited ligation more than those at the first position 3′ to the nick. The mismatches at other positions 5′ to the nick (3rd to 7th sites) exhibited less inhibition on ligation. However, the introduction of a C/C mismatch at the third position 5′ to the nick could completely inhibit the ligation of the terminal-mismatched nick of an oligonucleotide duplex by M. jannaschii DNA ligase. Therefore, introducing an additional mismatch at the third position 5′ to the SNP site is a more effective approach in genotyping by M. jannaschii DNA ligase.     

Structural Requirements forFokI-DNA Interaction and Oligodeoxyribonucleotide-instructed Cleavage

TheFokI restriction endonuclease recognizes the double-stranded (ds) 5′-GGATG-3′ site and cuts at the 9th and 13th nucleotides downstream from the 5′-3′ and 3′-5′ strands, respectively. To elucidate the interaction betweenFokI and DNA, and the effect of Mg²⁺on this interaction, we usedFokI with various combinations of dsDNA, single-stranded (ss) DNA and oligodeoxyribonucleotides (oligos) containing a double-stranded hairpin carrying theFokI recognition site. Oligo- and dsDNA-FokI interactions showed that for fully effective recognition, two or more base-pairs were required outside the 5′-GGATG-3′ site. When usingFokI with ssDNA and oligos, precise cutting with no observable byproducts was observed at the 9th or 13th nucleotide. This was independent of whether the region between the recognition and cut sites was perfectly complementary or whether there were up to four mismatches in this region, or a single mismatch within the cut site. Moreover,FokI cleavage, when followed by step-wise filling-in ofFokI cohesive ends in the dsDNA, allowedFokI to recleave such sites when two or more nucleotides were added, releasing 2-mer, 3-mer, or 4-mer single-stranded chains. Electrophoretic mobility shift assays showed that the DNA helix was bent when complexed withFokI (without Mg²⁺). Such a complex, when formed in the absence of Mg²⁺, did not accept the subsequently added Mg²⁺for several minutes. This suggests a tight, diffusion-resistant contact between the enzyme and the cognate DNA sequence. In the presence of Mg²⁺, the half-life of the complexFokI and dsDNA was 12 minutes at 22°C. In the absence of Mg²⁺, such a complex, possessing a terminally located 5′-GGATG-3′ site, had a half-life of 1.5 to 2 minutes. However, if magnesium ions were present, this complex had a stability similar to that of a complex formed with dsDNA containing a centrally located 5′-GGATG-3′ site.     

Evidence for the presence of chromatin-bound deoxyribonucleic acid phosphatase-exonuclease in Yoshida sarcoma ascites cells

An enzyme which cleaves the phosphoester bond of 3′-phosphoryl termini of DNA was isolated and purified from the chromatin of Yoshida sarcoma cells. The DNA phosphatase is specific for only 3′-phosphorylated DNA with a lesser activity for its single stranded form. Phosphoester bonds of various nucleotides, 3′-phosphorylated RNA and 5′-phosphorylated DNA were not hydrolysed by the enzyme. The DNA phosphatase required 10 mM MgCl₂, and was inactivated by 70 % with 1 mM ?-chloromercuribenzoate and completely by heat treatment at 70° for 5 min. Furthermore, an exonuclease activity could not be separated from the purified DNA phosphatase.