DNA-cleaving Enzymes Research Articles

A new method for determining the stereochemistry of DNA cleavage reactions: application to the SfiI and HpaII restriction endonucleases and to the MuA transposase.

A new method was developed for tracking the stereochemical path of enzymatic cleavage of DNA. DNA with a phosphorothioate of known chirality at the scissile bond is cleaved by the enzyme in H218O. The cleavage produces a DNA molecule with the 5'-[16O,18O, S]-thiophosphoryl group, whose chirality depends on whether the cleavage reaction proceeds by a single-step hydrolysis mechanism or by a two-step mechanism involving a protein-DNA covalent intermediate. To determine this chirality, the cleaved DNA is joined to an oligonucleotide by DNA ligase. Given the strict stereochemistry of the DNA ligase reaction, determined here, the original chirality of the phosphorothioate dictates whether the 18O is retained or lost in the ligation product, which can be determined by mass spectrometry. This method has advantages over previous methods in that it is not restricted to particular DNA sequences, requires substantially less material, and avoids purification of the products at intermediate stages in the procedure. The method was validated by confirming that DNA cleavage by the EcoRI restriction endonuclease causes inversion of configuration at the scissile phosphate. It was then applied to the reactions of the SfiI and HpaII endonucleases and the MuA transposase. In all three cases, DNA cleavage proceeded with inversion of configuration, indicating direct hydrolysis of the phosphodiester bond by water as opposed to a reaction involving a covalent enzyme-DNA intermediate.

Techniques for single molecule sequencing

A method is described that demonstrates a new technique for rapid and high-throughput single molecule sequencing. This sequencing technique is based on the successive enzymatic degradation of fluorescently labeled single DNA molecules, and the detection and identification of the released monomer molecules according to their sequential order in a microstructured channel. The detection technique is evolved from confocal fluorescence microscopy, with two different laser sources to excite the individual mononucleotides that are either labeled with tetramethylrhodamine (TMR) or Cyanine5 (Cy5). The handling of DNA which is immobilized on carrier beads, and the detection of the cleaved monomers is performed in optically transparent and biochemically inert microstructures (glass or PMMA) with detection channels of 7 μ × 10 μm. The projected rate of sequencing is ≈100 bases min−1, dependent solely on the rate of the enzymatic DNA cleavage.

Genomic footprinting of budding yeast replication origins during the cell cycle

Publisher Summary This chapter discusses the genomic footprinting of budding yeast replication origins during the cell cycle. Nucleotide resolution genomic footprinting can be a powerful tool for the monitoring of protein-DNA interactions in vivo or in a cell extract. The technique involves the use of an enzymatic or chemical agent as a probe to induce modifications in the DNA as the first step. The presence of a protein bound to the DNA can alter the specificity of the probe, either reducing the level of modification (implying protection) or increasing the level of modification (resulting in hypersensitive sites). These alterations can include chemical or enzymatic cleavage of DNA or chemical modifications of the DNA. A number of genomic footprinting procedures have been described; however, the use of this technique has often been limited by problems encountered in the detection of signals derived from sequences that represent only a small fraction of the total DNA, such as single-copy sequences in the eukaryotic genome.

Site-specific restriction endonucleases in Bacillus licheniformis

We systematically studied site-specific restriction endonucleases in Bacillus licheniformis strains and detected endonuclease activity in 25 of 217 strains tested. Three different activities were obtained. One of these activities detected in 21 strains was the most representative within the species and produced a banding pattern, after digestion of A DNA, identical to that seen with ClaI. Two other strains isolated from soil samples from China and USA were found to produce a DNA-cleaving enzyme with the same recognition sequence as BsaI. One producer strain, isolated from a Peruvian soil sample, showed to possess a mixture of two isoschizomers, ClaI and BsaI. Finally, one strain produced an endonuclease activity, not previously described in B. licheniformis, that showed the same recognition sites as Bsu361.

Site-specific restriction endonucleases in Bacillus licheniformis

We systematically studied site-specific restriction endonucleases in Bacillus licheniformis strains and detected endonuclease activity in 25 of 217 strains tested. Three different activities were obtained. One of these activities detected in 21 strains was the most representative within the species and produced a banding pattern, after digestion of A DNA, identical to that seen with ClaI. Two other strains isolated from soil samples from China and USA were found to produce a DNA-cleaving enzyme with the same recognition sequence as BsaI. One producer strain, isolated from a Peruvian soil sample, showed to possess a mixture of two isoschizomers, ClaI and BsaI. Finally, one strain produced an endonuclease activity, not previously described in B. licheniformis, that showed the same recognition sites as Bsu361.

Evidence for an evolutionary relationship among type-II restriction endonucleases

Type-II restriction-modification (R-M) systems comprise two enzymes, a DNA methyltransferase (MTase) and a restriction endonuclease (ENase), each of which specifically interact with the same 4–8-bp sequence. All type-II MTases share several amino acid (aa) sequence motifs, which makes an evolutionary relatedness among these enzymes probable. The type-II ENases, in contrast, except for some homologous isoschizomers, do not share significant aa sequence similarity. Therefore, ENases in general have been considered unrelated. Here we show that in addition to the analysis of the genotype (aa sequence), a comparison of the phenotype (recognition sequence) of these enzymes can provide independent information regarding evolutionary relationships, and thereby, help to analyze the significance of weak aa sequence similarities. Multistep Monte-Carlo analyses were employed to demonstrate that the recognition sequences of those ENases, which were found to be related by a progressive multiple aa sequence alignment, are more similar to each other than would be expected by chance. This analysis supports the notion that not only type-II MTases, but also type-II ENases did not arise independently in evolution, but rather evolved from one or a few primordial DNA-modifying and DNA-cleaving enzymes, respectively.

SruI restriction endonuclease from Selenomonas ruminantium.

SruI, specific restriction endonuclease, has been characterized from Selenomonas ruminantium isolated from the rumen of fallow deer. Results from the study demonstrate that S. ruminantium 18D possesses a type II restriction endonuclease, which recognizes the sequence 5'-TTT decreases AAA-3'. The recognition sequence of SruI was identified using digestions on pBR322, pBR328, pUC18, M13mp18RF, pACYC184 and lambda DNA. The cleavage patterns obtained were compared with computer-derived data. SruI recognises the palindromic hexanucleotide sequence and cleaves DNA after the third T in the sequence, producing blunt ends. The purification and characterization of restriction endonuclease SruI presented here is the first described for Selenomonas ruminantium spp. and demonstrates that this microorganism possesses a DNA-cleaving enzyme with the same specificity as DraI or AhaIII.

SruI restriction endonuclease fromSelenomonas ruminantium

SruI, specific restriction endonuclease, has been characterized from Selenomonas ruminantium isolated from the rumen of fallow deer. Results from the study demonstrate that S. ruminantium 18D possesses a type II restriction endonuclease, which recognizes the sequence 5'-TTT decreases AAA-3'. The recognition sequence of SruI was identified using digestions on pBR322, pBR328, pUC18, M13mp18RF, pACYC184 and lambda DNA. The cleavage patterns obtained were compared with computer-derived data. SruI recognises the palindromic hexanucleotide sequence and cleaves DNA after the third T in the sequence, producing blunt ends. The purification and characterization of restriction endonuclease SruI presented here is the first described for Selenomonas ruminantium spp. and demonstrates that this microorganism possesses a DNA-cleaving enzyme with the same specificity as DraI or AhaIII.

T-cell receptor alpha chain germline gene polymorphisms in multiple sclerosis.

It has been proposed that genetic predisposition to multiple sclerosis (MS) and other diseases with autoimmune features is conferred by T-cell receptor (TcR) genes in addition to HLA class II genes. Although a family study has suggested linkage of susceptibility to MS to TcR genes, reports of disease associations with restriction fragment length polymorphism (RFLP)-defined alleles of TcR genes have been difficult to confirm, including a report of association of MS with TcR beta-chain gene RFLPs. We report here the distribution of three sets of RFLPs of the TcR alpha chain genes. Similar frequencies in patients and controls were observed for TaqI and BglII RFLPs of the TcR alpha constant segment (C alpha), the latter earlier reported to be associated with MS. A previously reported MS-associated PssI RFLP of the V alpha 12 and C alpha gene segments could not be confirmed. Our results indicate that these seemingly polymorphic restriction fragments are possibly the results of incomplete enzymatic cleavage of DNA in the RFLP analysis. We conclude that no convincing evidence exists for the association of MS with RFLPs of the TcR alpha or beta chain genes.

Assessment of DNA binding of platinum-radiosensitizer complexes by inhibition of restriction enzymes

A simple and rapid method has been used to compare the binding of platinum complexes to DNA, in a relatively qualitative manner. A compound bound at or near the restriction site inhibits enzymatic cleavage of DNA; inhibition of BamHI and EcoRI activity by complexes was assessed in this study using linearized pSV2-gpt plasmid. Our particular interest was in DNA binding by complexes of platinum (Pt) with known organic radiosensitizers (RS), to determine whether the Pt was able to target the RS to the DNA. Although the Pt-RS complexes investigated themselves have moderate radiosensitizing ability (like the inorganic complexes, cis- or trans-diamminedichloroplatinum(II), c- or t-DDP) none of the Pt-RS inhibit to the same extent as c- or t-DDP. However, there appears to be some correlation between enhanced radiosensitization by Pt-RS over Pt(RS) 2, with the degree of Pt binding (as assessed by our assay). Our results using isolated DNA suggest that not all complexes bind well (e.g. Pt with two RS ligands), but that in certain cases (e.g. Pt with only one RS), it is possible to target the drug to the DNA. An ammine or amine ligand may be required in order to target a radiosensitizer to DNA using platinum.

Route of herpesvirus spread traced with aid of DNA-cleaving enzymes

DNA restriction endonucleases, enzymes that cleave DNA at specific sites, have been talked about primarily in connection with recombinant DNA technology. Surprisingly, then, one of their first medical applications is not in the synthesis of insulin or human growth hormone, but as a tool for investigating the epidemiology of herpesvirus outbreaks in hospitals. Restriction endonucleases split DNA at specific nucleotide sequences, yielding each time the same unique set of DNA fragments from a given DNA. If the DNA is radioactively labeled, the fragments may be separated on an agarose slab in an electric field and visualized by exposing the slab to roentgenographic film (figure, next page). Analysis of DNA from more than 80 different strains of herpesvirus in the laboratory of Bernard Roizman, ScD, professor of microbiology at the University of Chicago, has shown that any two epidemiologically distinct strains of herpesvirus can be distinguished by the fragments produced from

Route of herpesvirus spread traced with aid of DNA-cleaving enzymes

Route of herpesvirus spread traced with aid of DNA-cleaving enzymes