Coincidence of cleavage sites of intron endonuclease I-TevI and critical sequences of the host thymidylate synthase gene

To maximize spread of their host intron or intein, many homing endonucleases recognize nucleotides that code for important and conserved amino acid residues of the target gene. Here, we examine the cleavage requirements for I-TevI, which binds a stretch of thymidylate synthase (TS) DNA that codes for functionally critical residues in the TS active site. Using an in vitro selection scheme, we identified two base-pairs in the I-TevI cleavage site region as important for cleavage efficiency. These were confirmed by comparison of I-TevI cleavage efficiencies on mutant and on wild-type substrates. We also showed that nicking of the bottom strand by I-TevI is not affected by mutation of residues surrounding the bottom-strand cleavage site, unlike other homing endonucleases. One of these two base-pairs is universally conserved in all TS sequences, and is identical with a previously identified cleavage determinant of I-BmoI, a related GIY-YIG endonuclease that binds a homologous stretch of TS-encoding DNA. The other base-pair is conserved only in a subset of TS genes that includes the I-TevI, but not the I-BmoI, target sequence. Both the I-TevI and I-BmoI cleavage site requirements correspond to functionally critical residues involved in an extensive hydrogen bond network within the TS active site. Remarkably, these cleavage requirements correlate with TS phylogeny in bacteria, suggesting that each endonuclease has individually adapted to efficiently cleave distinct TS substrates.     

DNA binding and cleavage by the HNH homing endonuclease I-HmuI

The structure of I-HmuI, which represents the last family of homing endonucleases without a defining crystallographic structure, has been determined in complex with its DNA target. A series of diverse protein structural domains and motifs, contacting sequential stretches of nucleotide bases, are distributed along the DNA target. I-HmuI contains an N-terminal domain with a DNA-binding surface found in the I-PpoI homing endonuclease and an associated HNH/N active site found in the bacterial colicins, and a C-terminal DNA-binding domain previously observed in the I-TevI homing endonuclease. The combination and exchange of these features between protein families indicates that the genetic mobility associated with homing endonucleases extends to the level of independent structural domains. I-HmuI provides an unambiguous structural connection between the His-Cys box endonucleases and the bacterial colicins, supporting the hypothesis that these enzymes diverged from a common ancestral nuclease.     

Flexible DNA target site recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI

Homing endonucleases are highly specific catalysts of DNA strand breaks that induce the transposition of mobile intervening sequences containing the endonuclease open reading frame. These enzymes recognize long DNA targets while tolerating individual sequence polymorphisms within those sites. Sequences of the homing endonucleases themselves diversify to a great extent after founding intron invasion events, generating highly divergent enzymes that recognize similar target sequences. Here, we visualize the mechanism of flexible DNA recognition and the pattern of structural divergence displayed by two homing endonuclease isoschizomers. We determined structures of I-CreI bound to two DNA target sites that differ at eight of 22 base-pairs, and the structure of an isoschizomer, I-MsoI, bound to a nearly identical DNA target site. This study illustrates several principles governing promiscuous base-pair recognition by DNA-binding proteins, and demonstrates that the isoschizomers display strikingly different protein/DNA contacts. The structures allow us to determine the information content at individual positions in the binding site as a function of the distribution of direct and water-mediated contacts to nucleotide bases, and provide an evolutionary snapshot of endonucleases at an early stage of divergence in their target specificity.     

Cross-linking of SsoII restriction endonuclease to cognate and non-cognate DNAs

Specific and non-specific interactions SsoII restriction endonuclease (R·SsoII) were probed by the method of covalent attachment to modified DNA containing an active monosubstituted pyrophosphate internucleotide bond instead of a phosphodiester one. R·SsoII with six N-terminal His residues was shown to be cross-linked to duplexes with this type of modification, either containing or not the recognition sequence. Competition experiments with covalent attachment of R·SsoII to activated DNAs demonstrated the similar affinity of the enzyme to cognate and non-cognate DNAs in the absence of cofactor, Mg²⁺ ions.     

Crystallization and preliminary X-ray studies of I-PpoI: a nuclear, intron-encoded homing endonuclease from Physarum polycephalum.

The homing endonuclease I-PpoI is encoded by an optional third intron, Pp LSU 3, found in nuclear, extrachromosomal copies of the Physarum polycephalum 26S rRNA gene. This endonuclease promotes the lateral transfer or "homing" of its encoding intron by recognizing and cleaving a partially symmetric, 15 bp homing site in 26S rDNA alleles that lack the Pp LSU 3 intron. The open reading frame encoding I-PpoI has been subcloned, and the endonuclease has been overproduced in E. coli. Purified recombinant I-PpoI has been co-crystallized with a 21 bp homing site DNA duplex. The crystals belong to space group P3(1)21, with unit cell dimensions a = b = 114 A, c = 89 A. The results of initial X-ray diffraction experiments indicate that the asymmetric unit contains an enzyme homodimer and one duplex DNA molecule, and that the unit cell has a specific volume of 3.4 A3/dalton. These experiments also provide strong evidence that I-PpoI contains several bound zinc ions as part of its structure.     

Metal ion binding in the active site of the junction-resolving enzyme T7 endonuclease I in the presence and in the absence of DNA

Endonuclease I of bacteriophage T7 is a DNA junction-resolving enzyme. We have previously used crystallography to demonstrate the binding of two manganese ions into the active site that is formed by three carboxylate (Glu 20, Asp 55 and Glu 65) and a lysine residue (Lys 67). Endonuclease I is active in the presence of magnesium, manganese, iron (II) and cobalt (II) ions, weakly active in the presence of nickel, copper (II) and zinc ions, and completely inactive in the presence of calcium ions. However, using calorimetry, we have observed the binding of two calcium ions to the free enzyme in a manner very similar to the binding of manganese ions. In the presence of iron (II) ions, we have obtained a cleavage of the continuous strands of a junction bound by endonuclease I, at sites close to (but not identical with) enzyme-induced hydrolysis. The results suggest that this arises from attack by locally generated hydroxyl radicals, arising from iron (II) ions bound into the active site. This therefore provides an indirect way of examining metal ion binding in the enzyme-junction complex. Ion binding in free protein (by calorimetry) and the enzyme-junction complex (iron-induced cleavage) have been studied in series of active-site mutants. Both confirm the importance of the three carboxylate ligands, and the lack of a requirement for Lys67 for the ion binding. Calorimetry points to particularly critical role of Asp55, as mutation completely abolishes all binding of both manganese and calcium ions.     

Assessing the plasticity of DNA target site recognition of the PI-SceI homing endonuclease using a bacterial two-hybrid selection system

The PI-SceI protein from Saccharomyces cerevisiae is a member of the LAGLIDADG family of homing endonucleases that have been used in genomic engineering. To assess the flexibility of the PI-SceI-binding interaction and to make progress towards the directed evolution of homing endonucleases that cleave specified DNA targets, we applied a two-hybrid method to select PI-SceI variants from a randomized expression library that bind to different DNA substrates. In particular, the codon for Arg94, which is located in the protein splicing domain and makes essential contacts to two adjacent base-pairs, and the codons for four proximal residues were randomized. There is little conservation of the wild-type amino acid residues at the five randomized positions in the variants that were selected to bind to the wild-type site, yet one of the purified derivatives displays DNA-binding specificity and DNA endonuclease activity that is similar to that of the wild-type enzyme. A spectrum of DNA-binding behaviors ranging from partial relaxation of specificity to marked shifts in target site recognition are present in variants selected to bind to sites containing mutations at the two base-pairs. Our results illustrate the inherent plasticity of the PI-SceI/DNA interface and demonstrate that selection based on DNA binding is an effective means of altering the DNA cleavage specificity of homing endonucleases. Furthermore, it is apparent that homing endonuclease target specificity derives, in part, from constraints on the flexibility of DNA contacts imposed by hydrogen bonds to proximal residues.     

Identification of a nuclear protein interacting with a novel site on rat androgen receptor promoter after transcription factor NFkB is displaced from adjacent site

Sequence-specific DNA-protein interactions mediate the regulation of rat androgen receptor (rAR) gene expression. Previously, DNase I footprinting revealed that nuclear factor kappa B (NFkB) binds to region -574 to -554 on rAR promoter and represses its expression. In this study, we demonstrate that when NFkB protein is removed from its site by competitor DNA in DNase I footprinting reaction, a new DNase I protected region is formed overlapping adjacently (-594 to -561). This indicates that another nuclear protein (named here as FRN, factor repressed by NFkB) binds to rAR promoter only after NFkB protein is displaced. By competitive electrophoretic mobility shift assay and mutation analysis, we confirmed the formation of FRN-DNA complex. FRN interacts with a novel sequence on rAR promoter and may play a role in regulation of rAR gene expression in concert with NFkB.     

Purification and properties of a site-specific restriction endonuclease AaaI from Acetobacter aceti subsp. aceti No. 1023

Abstract A type II restriction endonuclease, named Aaa I, was purified from Acetobacter aceti subsp. aceti No. 1023. The optimum pH and temperature were determined to be 8.5 and 37°C, respectively. The enzyme activity was stimulated by the addition of either NaCl or KCl and their optimum concentrations were 100 mM for both cations. Aaa I recognized the hexanucleotide sequence and cleaved it at the positions indicated by the arrows. Aaa I was an isoschizomer of Xma III from Xanthomonas malvacaerum and Eco 52I from Escherichia coli .     

Purification and characterization of restriction endonuclease BcoI from a soil isolate of Bacillus coagulans

Abstract A soil identified as Bacillus coagulans is found to produce Bco I, an isoschizomer of Ava I and with the same cleavage site. This thermal stable enzyme, Bco I, is produced at high level and can be isolated by passing the crude bacterial lysate through a DEAE-cellulose column.     

Nickase and a protein encoded by an open reading frame downstream from the nickase BspD6I gene form a restriction endonuclease complex

We are the first to have isolated a protein (186 amino acid residues) encoded by the open reading frame adjacent to the end of the BspD6I nickase (N.BspD6I) gene. Cleavage of both DNA strands near the sequence recognized by nickase (5 -GAGTC/5 -GACTC) occurs when this protein is added to the reaction mixture containing N.BspD6I. The protein encoded by the open reading frame and the nickase are suggested to be subunits of heterodimeric restriction endonuclease R.BspD6I.     

Morphological and genetic analysis of three bacteriophages of Serratia marcescens isolated from environmental water

Increases in multidrug-resistant strains of Serratia marcescens are of great concern in pediatrics, especially in neonatal intensive care units. In the search for bacteriophages to control infectious diseases caused by multidrug-resistant S. marcescens , three phages (KSP20, KSP90, and KSP100) were isolated from environmental water and were characterized morphologically and genetically. KSP20 and KSP90 belonged to morphotype A1 of the family Myoviridae , and KSP100 belonged to morphotype C3 of the family Podoviridae . Analysis of the DNA region coding virion proteins, together with their morphological features, indicated that KSP20, KSP90, and KSP100 were related to the P2-like phage (temperate), T4-type phage (virulent), and phiEco32 phage (virulent), respectively. Based on amino acid sequences of the major capsid protein, KSP90 formed a new branch with a Stenotrophomonas maltophilia phage, Smp14, in the T4-type phage phylogeny. Both Smp14 and phiEco32 have been reported as potential therapeutic phages. These results suggest that KSP90 and KSP100 may be candidate therapeutic phages to control S. marcescens infection.     

Isolation and characterization of a type II restriction endonuclease from Streptococcus thermophilus

The alpha-toxin (phospholipase C) of Clostridium perfringens has been reported to contain catalytically essential zinc ions. We report here that histidine residues are essential for the co-ordination of these ion(s). Incubation of alpha toxin with diethylpyrocarbonate, a histidine modifying reagent, did not result in the loss of phospholipase C activity unless the protein was first incubated with EDTA, suggesting that zinc ions normally protect the susceptible histidine residues. When the amino acid sequences of three phospholipase C's were aligned, essential zinc binding histidine residues in the non-toxic B. cereus phospholipase C were found in similar positions in the toxic C. perfringens enzyme and the weakly toxic C. bifermentans phospholipase C.     

Bvui: a site-specific endonuclease from Bacillus vulgatis

A site-specific endodeoxyribonuclease was partially purified from an extract of osmotically shocked spheroplasts of a Bacillus vulgatis strain; the enzyme has been designated BvuI and its activity was characterized. The locations of BvuI-generated cleavages on bacteriophage lambda and M13 derivatives mp7, mp8 and mp9, SV40 and PBR322 DNA molecules were determined. BvuI was shown to recognize the DNA sequence decreases 5'-G-Pu-G-C-Py-C-3' 3'-C-Py-C-G-Pu-G-5' increases and cleaves it at the positions indicated by arrows. Two identical BvuI recognition sites separated by fourteen nucleotide pairs were shown to occur within the tetracycline resistance gene of PBR322; BvuI should be useful for molecular cloning in that plasmid vector.     

Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection

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