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Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array.sup.1. Spacer insertion is carried out by the Cas1-Cas2 integrase complex.sup.2-4. A substantial fraction of CRISPR-Cas systems use a Fe-S cluster containing Cas4 nuclease to ensure that spacers are acquired from DNA flanked by a protospacer adjacent motif (PAM).sup.5,6 and inserted into the CRISPR array unidirectionally, so that the transcribed CRISPR RNA can guide target searching in a PAM-dependent manner. Here we provide a high-resolution mechanistic explanation for the Cas4-assisted PAM selection, spacer biogenesis and directional integration by type I-G CRISPR in Geobacter sulfurreducens, in which Cas4 is naturally fused with Cas1, forming Cas4/Cas1. During biogenesis, only DNA duplexes possessing a PAM-embedded 3'-overhang trigger Cas4/Cas1-Cas2 assembly. During this process, the PAM overhang is specifically recognized and sequestered, but is not cleaved by Cas4. This 'molecular constipation' prevents the PAM-side prespacer from participating in integration. Lacking such sequestration, the non-PAM overhang is trimmed by host nucleases and integrated to the leader-side CRISPR repeat. Half-integration subsequently triggers PAM cleavage and Cas4 dissociation, allowing spacer-side integration. Overall, the intricate molecular interaction between Cas4 and Cas1-Cas2 selects PAM-containing prespacers for integration and couples the timing of PAM processing with the stepwise integration to establish directionality. Structures of the Cas4-Cas1-Cas2 complex from Geobacter sulfurreducens show that a 3'-overhang in the protospacer adjacent motif is required for complex assembly and spacer insertion into the CRISPR array., Author(s): Chunyi Hu [sup.1] , Cristóbal Almendros [sup.2] [sup.3] , Ki Hyun Nam [sup.4] , Ana Rita Costa [sup.2] [sup.3] , Jochem N. A. Vink [sup.2] [sup.3] , Anna C. [...]

Cleavage of viral DNA by the bacterial Type III Restriction-Modification enzymes requires the ATP-dependent long-range communication between a distant pair of DNA recognition sequences, The classical view is that Type III endonuclease activity is only activated by a pair of asymmetric sites in a specific head-to-head inverted repeat. Based on this assumption and due to the presence of helicase domains in Type III enzymes, various motor-driven DNA translocation models for communication have been suggested. Using both single-molecule and ensemble assays we demonstrate that Type III enzymes can also cleave DNA with sites in tail-to-tail repeat with high efficiency. The ability to distinguish both inverted repeat substrates from direct repeat substrates in a manner independent of DNA topology or accessory proteins can only be reconciled with an alternative sliding mode of communication, diffusion | helicase | motor | switch doi: 10. 073/pnas.1001637107

Homing endonuclease genes (HEGs) encode proteins that in the heterozygous state cause double-strand breaks in the homologous chromosome at the precise position opposite the HEG. If the double-strand break is repaired using the homologous chromosome, the HEG becomes homozygous, and this represents a powerful genetic drive mechanism that might be used as a tool in managing vector or pest populations. HEGs may be used to decrease population fitness to drive down population densities (possibly causing local extinction) or, in disease vectors, to knock out a gene required for pathogen transmission. The relative advantages of HEGs that target viability or fecundity, that are active in one sex or both, and whose target is expressed before or after homing are explored. The conditions under which escape mutants arise are also analyzed. A different strategy is to place HEGs on the Y chromosome that cause one or more breaks on the X chromosome and so disrupt sex ratio. This strategy can cause severe sex-ratio biases with efficiencies that depend on the details of sperm competition and zygote mortality. This strategy is probably less susceptible to escape mutants, especially when multiple X shredders are used.

A genome sequence of honeybee Apis mellifera has shown it to be the first insect with a full complement of DNA methyltransferases which are catalytically active and that Apis genes can be methylated in specific patterns. These results establish bees as a model to analyze the function of DNA methylation system in invertebrate organisms and might also be helpful in understanding evolutionary aspects of DNA methylation in higher eukaryotes.

Among all restriction endonucleases known to date, Bfil is unique in cleaving DNA in the absence of metal ions. Bfil represents a different evolutionary lineage of restriction enzymes, as shown by its crystal structure at 1.9-[Angstrom] resolution. The protein consists of two structural domains. The N-terminal catalytic domain is similar to Nuc, an EDTA-resistant nuclease from the phospholipase D superfamily. The C-terminal DNA-binding domain of Bfil exhibits a [beta]-barrel-like structure very similar to the effector DNA-binding domain of the [Mg.sup.2+]-dependent restriction enzyme EcoRII and to the B3-like DNA-binding domain of plant transcription factors. Bfil presumably evolved through domain fusion of a DNA-recognition element to a nonspecific nuclease akin to Nuc and elaborated a mechanism to limit DNA cleavage to a single double-strand break near the specific recognition sequence. The crystal structure suggests that the interdomain linker may act as an autoinhibitor controlling Bfil catalytic activity in the absence of a specific DNA sequence. A PSI-BLAST search identified a Bfil homologue in a Mesorhizobium sp. BNC1 bacteria strain, a plant symbiont isolated from an EDTA-rich environment. restriction endonuclease | x-ray crystallography

A group of new DNA enzymes that possess a synchronized RNA-cleavage flourescence-signaling ability and exhibit wide-ranging metal-ion and pH dependences is reported. The demonstration of DNA enzymes that are functional under extremely high acidity indicates that DNA has the ability to perform efficient catalysts even under harsh reaction conditions.

Unlike other restriction enzymes, Bfil functions without metal ions. It recognizes an asymmetric DNA sequence, 5'-ACTGGG-3', and cuts top and bottom strands at fixed positions downstream of this sequence. Many restriction enzymes are dimers of identical sub-units, with one active site for each DNA strand. Others, like Fokl, dimerize transiently during catalysis. Bfil is also a dimer but it has only one active site, at the dimer interface. We show here that Bfil remains a dimer as it makes double-strand breaks in DNA and that its single active site acts sequentially, first on the bottom and then the top strand. Hence, after cutting the bottom strand, a rearrangement of either the protein and/or the DNA in the Bfil-DNA complex must switch the active site to the top strand. Low pH values selectively block top-strand cleavage, converting Bfil into a nicking enzyme that cleaves only the bottom strand. The switch to the top strand may depend on the ionization of the cleaved 5' phosphate in the bottom strand. Bill thus uses a mechanism for making double-strand breaks that is novel among restriction enzymes.

Byline: E. P. Delver (1), M. V. Sobennikova (2), N. G. Belogurova (2), S. V. Nekrasov (2), M. D. Kolesnikova (2), O. V. Agafonova (1), A. A. Belogurov (1) Keywords: DNA restriction; antirestriction proteins; self-transmissible plasmids Abstract: A study was made of the functional role of the ArdA antirestriction motif (130-LLADVPETVALYFD-143) conserved among all known Ard (alleviation of restriction of DNA) proteins, which are encoded by self-transmissible plasmids and specifically inhibit type I restriction--modification systems. Conserved residues of the motif were individually changed, and the resulting mutants tested for in vivo activity. Hydrophobic L130, L131, and V138 were substituted with negatively charged E negatively charged D133, E136, and D143 substituted with hydrophobic V and D127, D150, and D154 neighboring the antirestriction motif substituted with V. Four substitutions (L130E, L131E, V138E, and D143V) substantially (25--1000 times) reduced the ArdA activity. The other substitutions within or beyond the motif had no appreciable effect. Substitutions L130A and L131A each reduced the ArdA activity 10- to 20-fold, indicating that high hydrophobicity of L130 and L131 is important for the ArdA function. Thus, the antirestriction role of ArdA is indeed due to its conserved motif. Author Affiliation: (1) Institute of Experimental Cardiology, Cardiology Research Center, Ministry of Health of the Russian Federation, Moscow, 121552, Russia (2) Chemical Faculty, Moscow State University, Moscow, 117899, Russia Article History: Registration Date: 09/10/2004

The mutational analysis of the presumptive nucleolytic center of McrC subunit of the McrBC system of Escherichia coli suggests that McrC mediates DNA cleavage by McrBC. The catalytic center of the McrC is represented by the sequence TD(sup)224 ....D(sup)257 AK.

Research demonstrates that the DNA cleavage activity of the yeast homing endonuclease can be turned on and off with a redox switch. Data show that Cys-67/Cys-365 variants of the endonuclease exhibit different DNA binding conformations under redox conditions.The DNA binding defects reduce DNA cleavage activities of the disulfide containing proteins.

Research shows that PvuII endonuclease requires divalent metal ion for optimal DNA binding as indicated by the cognate affinity and sequence discrimination capacities in the presence of metal ions. The enzyme binds the target sequence with a K(sub)d of 50 picomoles in presence of calcium(II) as against 300 nanomoles in its absence.

The K(sub D) value of EcoRV restriction endonuclease for its GATATC target sequence ranges from 1.5 to 6.5 at pH 7.5 and 100 mM NaCl. When Ca(super 2+) is present, a decrease in pH facilitates the binding of specific sequences but not nonspecific sequences.

The tryptophan residue at position 104 of EcoRI endonuclease changes position upon DNA binding. The N-termini are essential for binding and catalysis and are located at the interface formed by loops created by residues 221-232.

Byline: S. M. Rastorguev (1), G. B. Zavilgelsky (1), K. Suzuki (2), K. Sakka (2) Keywords: antirestriction; ars operon; antirestriction motif Abstract: A study was made of the antirestriction activity of Acidiphilium multivorum AIU 301 ArsR, a repressor of the ars operon which confers resistance to arsenite and arsenate and is contained in pKW301. In Escherichia coli, arsR cloned under the control of P.sub.lac in a multicopy vector alleviated restriction of nonmodified [lambda] DNA by a factor of 120, six times more efficiently than its analogs of conjugal plasmids R64 (incI1) and R773 (incFI). Amino acid sequence analysis showed that the three ArsR proteins have a homologous region of 38 residues, including the antirestriction motif, in their N domains, whereas in the Ard proteins the motif is in the C domain. The other regions are nonhomologous, and pKW301 ArsR is 33 residues shorter than R64 and R773 ArsRs. The total charge is --4 in pKW301 ArsR and +2 in R64 and R733 ArsRs. A total negative charge was assumed to contribute to the antirestriction activity. Author Affiliation: (1) State Research Center GosNIIgenetika, Moscow, 113545, Russia (2) Laboratory of Applied Microbiology, Faculty of Bioresources, Mie University, Tsu, 514, Japan Article History: Registration Date: 08/10/2004

Mismatch repair in many organisms depends on three proteins: the mismatch-recognition protein MutS, a nicking endonuclease MutH, and MutL, which acts as a scaffold between these. However, many genomes lack MutL but possess MutS. In one of these cases, in a coral mitochondrial genome, a gene is present that encodes a MutS protein fused to an HNH nicking endonuclease, potentially eliminating the requirement for MutL. Likewise, many prokaryotes could operate similarly, independently of MutL by encoding a fused MutS-Smr (MutS2) protein. Smr, which is proposed to be a nicking endonuclease, can also be found separately in many eukaryotes, where it might play a role in mismatch repair or meiotic chromosome crossing-over.

Research has been conducted on the metal binding residues located at restriction endonuclease active sites. The use of Fenton chemistry techniques in identifying these residues is described.

Results demonstrate that interaction between DNA and the very short patch repair protein leads to an intercalation of three aromatic amino acid residues to DNA thereby dislocating base pair staking. The data point out to the structural basis for TG mismatch recognition in bacteria.

In the homing endonuclease I-PpoI, His98 activates a water molecule to begin the reaction, Arg61 and Asn119 stabilize the transition state and Asn119 also binds a Mg(sub)2+ ion that is essential for catalysis. Homing endonucleases cleave dsDNA in a site-specific manner.

Research was conducted to examine the role that the flap endonuclease, FEN1, plays in DNA replication and repair. The effect of flap modifications, inside and outside of the footprinted region was measured for their ability to influence the efficiency of flap cleavage. The objective was to determine whether the nuclease tracks from the 5'-end of the flap to the site of cleavage and whether such tracking involves threading the flap through a hole in the protein. Results indicate that some flap modifications can prevent or inhibit tracking but the tracking mechanism tolerates a variety of flap modifications.

ClpXP-dependent proteolysis has been implicated in the delayed detection of restriction activity after the acquisition of the genes (hsdR, hsdM, and hsdS) that specify EcoKI and EcoAI, representatives of two families of type I restriction and modification (R-M) systems. Modification, once established, has been assumed to provide adequate protection against a resident restriction system. However, unmodified targets may be generated in the DNA of an [hsd.sup.+] bacterium as the result of replication errors or recombination-dependent repair. We show that ClpXP-dependent regulation of the endonuclease activity enables bacteria that acquire unmodified chromosomal target sequences to survive. In such bacteria, HsdR, the polypeptide of the R-M complex essential for restriction but not modification, is degraded in the presence of ClpXP. A mutation that blocks only the modification activity of EcoKI, leaving the cell with [approximately equal to]600 unmodified targets, is not lethal provided that ClpXP is present. Our data support a model in which the HsdR component of a type I restriction endonuclease becomes a substrate for proteolysis after the endonuclease has bound to unmodified target sequences, but before completion of the pathway that would result in DNA breakage.

The biochemical role of the human DNA repair protein XPF has been studied by expressing and purifying the independent 104 kDa recombinant XPF protein from Escherichia coli. The XPF/ERCC1 protein complex is responsible for making one of the necessary incisions in eliminating a broad range of DNA adducts during nucleotide excision repair. Results indicate that XPF is an endonuclease and can bind DNA even when the ERCC1 subunit is not present. It has an N-terminal catalytic domain based on the endonuclease activity of a stable 70 kDa proteolysis fragment of XPF.

The presence of a metal cofactor was deemed vital in supporting the binding kinetics of TaqI endonuclease with its TCGA cognate sequence. This high-affinity specific binding interaction results in the formation of a highly stable TaqI-TCGA-Mg2+ complex with a 20-minute half life at 60 degrees celsius. However, in the absence of a metal cofactor, the TaqI enzyme tends to bind with all DNA sequences, including cognate site, star site and nonspecific site, which eventually leads to the formation of a binding site with a relatively the same magnitude of affinity.

Mutants of Gibberella fujikuroi that were deficient in gibberellin biosynthesis were induced by transformation-mediated mutagenesis with the vector pAN7-1. 24 GA-defective mutants were recovered in one of nine transformation experiments performed without the addition of a restriction enzyme. Each mutant had a similar Southern blot pattern, suggesting the integration of the vector into the same site. The addition of a restriction enzyme through mediated integration significantly increased the transformation rate and the rate of single-copy integration events.

A study was conducted to determine whether homopyrimidine oligo-RNA and oligo-2'-O-methyl-RNA containing 8-oxo-adenosine (AOH) and 8-oxo-O-methyl (AmOH) adenosine instead of cytidine can inhibit the sequence-specific cleavage of DNA binding proteins by the restriction endonuclease Ksp632-I. The simian virus 40 (SV40) DNA was used in the study. Results showed that the cleavage of SV40 DNA was inhibited by the AmOH-substituted 2'-O-mthyl RNA oligonucleotides but not by the homopyrimidine oligo-RNA and oligo-2'-O-methyl-RNA containing AOH moieties.

A study was conducted to investigate the genomic relationships between Entorococcus faecium strains from different sources and with varied levels of antibiotic resistance. Restriction endonuclease analysis was used to evaluate total chromosomal DNA of 47 strains. Results indicate that strain clusters can be formed according to host. The major groups of Entorococcus faecium strains were categorized under chicken, pork and human. It was also observed that strains from animals do not group with those of human's.

The divalent metal-dependent catalytic characteristics in phosphoryl transfer by EcoRV endonuclease were investigated by transient kinetic methods. The results of pre-steady-state measurements on short oligonucleotide substrates showed bursts of both Mg2+- and M2+-catalyzed DNA cleavage reactions. The results indicate that the product release step is partially or completely rate-limiting for for each metal ions. The steepness of the burst is greater for M2+ reactions and the overall rate is six-fold slower in the presence of this cofactor.

Research was conducted to examine the mutations that extend the specificity of the endonuclease activity of lambda terminase. The A-E515G, A-N509K and A-R504C mutations were isolated as suppressors of the cosN G2C C11G mutations. Results revealed by kinetic studies with purified terminase showed the nature of the cosN G2C C11G defect. Findings demonstrate that wild-type terminase has a reduced efficiency in cleaving cosN G2C C11G.

The hsd genes of Mycoplasma pulmonis encode restriction and modification enzymes exhibiting a high degree of sequence similarity to the type I enzymes of enteric bacteria. The S subunits of type I systems dictate the DNA sequence specificity of the holoenzyme and are required for both the restriction and the modification reactions. The M. pulmonis chromosome has two hsd loci, both of which contain two hsdS genes each and are complex, site-specific DNA inversion systems. Embedded within the coding region of each hsdS gene are a minimum of three sites at which DNA inversions occur to generate extensive amino acid sequence variations in the predicted S subunits. We show that the polymorphic hsdS genes produced by gene rearrangement encode a family of functional S subunits with differing DNA sequence specificities. In addition to creating polymorphisms in hsdS sequences, DNA inversions regulate the phase-variable production of restriction activity because the other genes required for restriction activity (hsdR and hsdM) are expressed only from loci that are oriented appropriately in the chromosome relative to the hsd promoter. These data cast doubt on the prevailing paradigms that restriction systems are either selfish or function to confer protection from invasion by foreign DNA.

The 2.15-[Angstrom] resolution cocrystal structure of EcoRV endonuclease mutant T93A complexed with DNA and [Ca.sup.2+] ions reveals two divalent metals hound in one of the active sites. One of these metals is ligated through an inner-sphere water molecule to the phosphate group located 3[prime] to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-Sp phosphoryl oxygen on the adjacent 3[prime] phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of EcoRV. EcoRI, PvuII, and BamHl endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous crystal structures, these data allow construction of a detailed model for the pretransition state configuration in EcoRV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.

An experiment was undertaken to locate the N-terminal region of the EcoRI endonuclease using site-directed fluorescence spectroscopy and chemical cross-linking. The N-termini of the endonuclease were believed to be involved in DNA cleavage and influenced the stability of the EcoRI-DNA complex without compromising specificity. Results revealed that the N-terminal region possessed a 16-residue sequence and that the N-termini were closer with each other at the dimer interface with constrained flexibility.

The structure and activity of the Pyrococcus furiosus's flap endonuclease 1 (FEN-1) were studied to determine its role in DNA repair and replication. The crystal structure of FEN-1 exhibited interactions for the single- and double-stranded portions of the flap DNA substrate. The binding of the flap DNA substrates' stranded regions were influenced by two structural motifs. It was revealed that the conserved C terminus of FEN-1 influenced its involvement in the DNA replication fork apparatus through the interaction with the C-terminal face of the proliferating cell nuclear antigen.

The thermostable restriction enzyme PspGI and the cloning and expression of the pspGIR and psrGIM genes in Escherichia coli were described. The modification genes of PspGI contained the conserved sequence motifs of alpha-aminomethyltransferases which suggested that it must be an N4-cytosine methylase. PspGI was expressed in E. coli via a T7 expression system. Recombinant PspGI had a half-life of two hours at 95 degrees C. PspGI may be used in DNA-based diagnostic applications.

A study described an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences followed by cleavage of the amplified material with restriction enzymes. The contributions of various ribotypes to total Bacteroides and Prevotella 16S rDNA were estimated followed by the estimation of the contribution of the sequences to total eubacterial 16S rDNA. The approach was found to be a rapid, convenient and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.

The DNA of the cyanobacterium Synechocystic sp. strain PCC 6803 was examined for modifications by methylation using restriction endonucleases. Sequence-specific methylations were detectable, while no restriction endonuclease activities were found. Changes in the DNA methylation pattern were detected, showing that the open reading frame slr0214 encodes a methyltransferase. The resulting protein in the subcloning and expression of slr0214 gene showed methyltransferase activity in vitro.

The restriction enzymes Asel and Dral were used to construct a physical and genetic map of the chromosome of the erythromycin-producing actinomycete Saccharopolyspora erythraea NRRL 2338. The digested fragments of the macrorestriction patterns of the restriction enzyme revealed a chromosome size of around 8Mb. It was also observed that 32% of the chromosome was covered by linking clones for the five contiguous Asel fragments. Moreover, the ribosomal RNA operons of S. erythraea contains an Asel site within the 16S(rrs) gene.

The diverse homing endonuclease proteins are encoded by genes with mobile and self-splicing introns. Four groups of homing endonucleases have been identified, and I-Ppol was the first identified His-Cys box homing endonuclease. Its structure is bound to homing-site DNA determined to a.8 A resolution. It has a new zinc-bound fold and endonuclease active site, and its structure is determined in uncleaved and cleaved complexes.

A systematic analysis of the sequence specificity of the four-way DNA junction-specific endonuclease CCE1 of Saccharomyces cerevisiae was undertaken through a single-turnover kinetic assay using synthetic DNA fragments. Results reveal that CCE1 discriminates the sequence 5'-ACTA and that the discrimination of the substrate takes place during the transition state. CCE1 exhibits conformational flexibility enabling the enzyme to cleave DNA at positions immediately adjacent to the usual cleavage site.

A study was conducted to examine the chemistry of glycosylases and endonucleases associated in base-excision repair. DNA repair enzymes need other enzymes to complete the DNA repair process. Alkylating agents associate with DNA to establish several modified bases while an array of enzymes maintain the DNA integrity and DNA repair mechanisms. DNA repair enzymes plays a major role in the cell that promotes a better understanding of various diseases such as cancer.

A study was conducted to test whether Saccharomyces cerevisiae supports two functional homologues of Escherichia coli endonuclease III. The E. coli strain DH5-alpha was utilized to subclone and propagate plasmids while yeast cells were exposed to 0.3 mM menadione. Results indicated S. cerevisiae possessed two functional homologues which differ from each other based on their amino acid sequences and inducibility by DNA damaging agents.

Analysis of the genetic changes in human tumors is often problematical because of the presence of normal stroma and the limited availability of pure tumor DNA. However, large amounts of highly reproducible 'representations' of tumor and normal genomes can be made by PCR from nanogram amounts of restriction endonuclease cleaved DNA that has been ligated to oligonucleotide adaptors. We show here that representations are useful for many types of genetic analyses, including measuring relative gene copy number, loss of heterozygosity, and comparative genomic hybridization. Representations may be prepared even from sorted nuclei from fixed and archived tumor biopsies.

Restriction endonucleases such as EcoRI bind and cleave DNA with great specificity and represent a paradigm for protein-DNA interactions and molecular recognition. Using osmotic pressure to induce water release, we demonstrate the participation of bound waters in the sequence discrimination of substrate DNA by EcoRI. Changes in solvation can play a critical role in directing sequence-specific DNA binding by EcoRI and are also crucial in assisting site discrimination during catalysis. By measuring the volume change for complex formation, we show that at the cognate sequence (GAATTC) EcoRI binding releases about 70 fewer water molecules than binding at an alternate DNA sequence (TAATTC), which differs by a single base pair. EcoRI complexation with nonspecific DNA releases substantially less water than either of these specific complexes. In cognate substrates (GAATTC) [k.sub.cat] decreases as osmotic pressure is increased, indicating the binding of about 30 water molecules accompanies the cleavage reaction. For the alternate substrate (TAATTC), release of about 40 water molecules accompanies the reaction, indicated by a dramatic acceleration of the rate when osmotic pressure is raised. These large differences in solvation effects demonstrate that water molecules can be key players in the molecular recognition process during both association and catalytic phases of the EcoRI reaction, acting to change the specificity of the enzyme. For both the protein-DNA complex and the transition state, there may be substantial conformational differences between cognate and alternate sites, accompanied by significant alterations in hydration and solvent accessibility.

Variants of the restriction enzyme EcoRV that depend on the flexibility of sequences flanking the recognition site for their cleavage activity were described via single-turnover cleavage studies with oligodeoxynucleotides and pre-steady-state and steady state cleavage experiments. The results suggested that the increased selectivity of the mutants may be caused by the catalytic phase of the reaction. The discriminatory abilities of the EcoRV variants may also be improved by choosing a suitable reaction environment, specifically low temperature and salt concentration.

The effect of linear diffusion on the mechanism of DNA cleavage by EcoRV was examined using Monte Carlo simulation. Linear diffusion effects was highest at 50 millimole NaCl under varying MgCl2 concentrations and increases in direct proportion to the Mg2+ concentrations up to 10 millimole. The comparison of the cleavage rates of linear DNA with either blocked or free ends revealed that EcoRV is not likely to be detached from the end of the linear DNA, since the enzyme merely bounces and resumes linear diffusion.

We report the characterization and partial purification of potato mitochondrial RNase Z, an endonuclease that generates mature tRNA 3[prime] ends. The enzyme consists of one (or more) protein(s) without RNA subunits. Products of the processing reaction are tRNA molecules with 3[prime] terminal hydroxyl groups and 3[prime] trailers with 5[prime] terminal phosphates. The main processing sites are located immediately 3[prime] to the discriminator and one nucleotide further downstream. This endonucleolytic processing at and close to the tRNA 3[prime] end in potato mitochondria suggests a higher similarity to the eukaryotic than to the prokaryotic tRNA 3[prime] processing pathway. Partial purification and separation of RNase Z from the 5[prime] processing activity RNase P allowed us to determine biochemical characteristics of the enzyme. The activity is stable over broad pH and temperature ranges, with peak activity at pH 8 and 30 [degrees] C. Optimal concentrations for [MgCl.sub.2] and KCl are 5 mM and 30 raM, respectively. The potato mitochondrial RNase Z accepts only tRNA precursors with mature 5[prime] ends. The precursor for [tRNA.sup.Phe] requires RNA editing for efficient processing by RNase Z.

A study was conducted to characterize three genes coding for the endonuclease, methylase and specificity subunits of a type I restriction-modification (R-M) system in the Lactococcus lactis plasmid pIL2614. The results indicate that this R-M system is probably of type IC based on factors such as plasmid location, sequence homologies and inactivation data.

A study is conducted to determine the existence of thermophilic Type II restriction endonuclease Taql in 152 isolates of the genus Thermus derived from hot springs located in four continents. 27 isolates were observed to contain isoschizomers of Taql. Six of these isolates were identified as Thermus species T. brockianus, T. scotoductus, T. thermophilus, T. filiformis and T. aquaticus. They vary with regard to thermal stability, isoelectric points, magnesium ion needs and subunit molecular masses.

We have developed and used a genetic selection system in Escherichia coli to study functional requirements for homing site recognition and cleavage by a representative eukaryotic mobile intron endonuclease. The homing endonuclease, I-CreI, was originally isolated from the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. I-CreI homing site mutants contained base pair substitutions or single base deletions that altered the rate of homing site cleavage and/or product release. I-CreI endonuclease mutants fell into six phenotypic classes that differed in in vivo activity, toxicity or genetic dominance. Inactivating mutations clustered in the N-terminal 60% of the I-CreI amino acid sequence, and two frameshift mutations were isolated that resulted in premature translation termination though retained partial activity. These mutations indicate that the N-terminal two-thirds of the I-CreI endonuclease is sufficient for homing site recognition and cleavage. Substitution mutations altered in four potential active site residues were examined: D20N, Q47H or R70A substitutions inactivated endonuclease activity, whereas S22A did not. The genetic approach we have taken complements phylogenetic and structural studies of mobile intron endonucleases and has provided new information on the mechanistic basis of I-CreI homing site recognition and cleavage.

Novel restriction enzymes can be created by fusing the nuclease domain of FokI endonuclease with defined DNA binding domains. Recently, we have characterized a domain (Z[Alpha]) from the N-terminal region of human double-stranded RNA adenosine deaminase (hADARI), which binds the Z-conformation with high specificity. Here we report creation of a conformation-specific endonuclease, Z[Alpha] nuclease, which is a chimera of Z[Alpha] and FokI nuclease. Purified Z[Alpha] nuclease cleaves negatively supercoiled plasmids only when they contain a Z-DNA forming insert, such as [(dC-dG).sub.(13)]. The precise location of the cleavage sites was determined by primer extension. Cutting has been mapped to the edge of the B-Z junction, suggesting that Z[Alpha] nuclease binds within the Z-DNA insert, but cleaves in the nearby B-DNA, by using a mechanism similar to type IIs restriction enzymes. These data show that Z[Alpha] binds Z-DNA in an environment similar to that in a cell. Z[Alpha] nuclease, a structure-specific restriction enzyme, may be a useful tool for further study of the biological role of Z-DNA.