The FokI methyltransferase from Flavobacterium okeanokoites. Purification and characterization of the enzyme and its truncated derivatives.

The gene encoding the FokI methyltransferase from Flavobacterium okeanokoites was cloned into an Escherichia coli vector. The transcriptional start sites were mapped as well as putative -10 and -35 regions of the fokIM promoter. Enzyme overproduction was ensured by cloning the fokIM gene under the phi 10 promoter of phase T7. M.FokI was purified using a two-step chromatography procedure. M.FokI is a monomeric protein with a M(r) = 76,000 +/- 1,500 under denaturing conditions. It contains 21 Arg residues, and at least one of which is required for activity as shown by inhibition using 2,3-butanedione. Deletion mutants in the N- and C-terminus of M.FokI were isolated and characterized. The N-terminal derivative (M.FokIN) methylates the adenine residue within the sequence 5'-GGATG-3', whereas the C-terminal derivative (M.FokIC) modifies the adenine residue within the sequence 5'-CATCC-3'. Substrate-protection studies, utilizing chemical modification combined with data on the effect of divalent cations and pH on methylation activity, proved the existence of two catalytic centers within the FokI methyltransferase molecule. M.FokI and its truncated derivatives require S-adenosyl-L-methionine as the methyl-group donor, and they are strongly inhibited by divalent cations (Mg2+, Ca2+, Ba2+, Mn2+, and Zn2+) and S-adenosyl-L-homocysteine. The Km values for the methyl donor, S-adenosyl-L-methionine are 0.6 microM (M.FokI), 0.4 microM (M.FokIN), and 0.9 microM (M.FokIC) while the Km values for substrate lambda DNA are 1.2 nM (M.FokI), 1.4 nM (M.FokIN), and 1.3 nM (M.FokIC).

Thermal cycle labeling: zeptomole detection sensitivity and microgram probe amplification using CviJl* restriction-generated oligonucleotides.

A new method for efficiently labeling and amplifying DNA probes from anonymous samples has been developed. The two/three base recognition endonuclease CviJI* restricts DNA to numerous small fragments primarily 20-60 bp in size. Thermal denaturation of these fragments results in sequence-specific oligonucleotides complementary to their cognate template. Repeated cycles of denaturation, annealing, and extension of such a multiprimed template by a thermostable DNA polymerase results in a significant amplification of the starting material. This method of amplification, referred to as thermal cycle labeling (TCL), appears to generate a large fraction of rearranged and presumably branched products. The inclusion of nucleotide analogs in the TCL reaction generates microgram amounts of haptentagged probe with a detection limit of 25 zmol (2.5 x 10(-20) mol). Reactions containing [alpha-33P]dCTP yield high-specific-activity probes (2.6 x 10(9) cpm/microgram) with reduced radiolytic decay and a useful shelf life of 1 month. CviJI* -generated primers circumvent the need for synthetic oligos while providing microgram amounts of amplified and labeled probes using the described TCL protocol.

Sequential amplification of cloned DNA as tandem multimers using class-IIS restriction enzymes.

In order to make high-copy-number multimers of DNA fragments in a tandem unit, two different gene amplification vectors (pSK9 and pBBS1) were developed. Two identical class-IIS restriction enzyme sites (BspMI for pSK9 and BbsI for pBBSI) were inversely oriented in each vector with the same cut site, creating asymmetric and complementary cohesive ends (5'-CCCC and 5'-GGGG). Multimers were made by: (i) cloning a target DNA into the class-IIS restriction enzyme cut site of each vector; (ii) excision of the monomeric insert by digestion with the class-IIS restriction enzyme; (iii) isolation of the fragments; (iv) self-ligation of the fragments; (v) cloning into the original vector digested with the class-IIS restriction enzyme; and (vi) repeating steps (i) through (v) to generate higher-order multimers. Various-sized multimers of a 93-bp DNA fragment encoding magainin, an antimicrobial peptide, were obtained with the gene amplification vector, pBBS1. Larger multimers, up to about 108 copies, were constructed from the monomer by the sequential amplification procedure. Of six different Escherichia coli hosts examined for the stability of multimers, the multimers were the most stable in E. coli D1210. The gene amplification vector system described here is very efficient and can be applied in the construction of tandem multimers of any kind of DNA, as long as the cloned DNA does not contain the cut site of the class-IIS restriction enzyme to be utilized.

Molecular cloning of the three base restriction endonuclease R.CviJI from eukaryotic Chlorella virus IL-3A.

R.CviJI is unique among site-specific restriction endonucleases in that its activity can be modulated to recognize either a two or three base sequence. Normally R.CviJI cleaves RGCY sites between the G and C to leave blunt ends. In the presence of ATP R.CviJI* cleaves RGCN and YGCY sites, but not YGCR sites. The gene encoding R.CviJI was cloned from the eukaryotic Chlorella virus IL-3A and expressed in Escherichia coli. The primary E.coli cviJIR gene product is a 278 amino acid protein initiated from a GTG codon, rather than the expected 358 amino acid protein initiated from an in-frame upstream ATG codon. Interestingly, the 278 amino acid protein displays the normal restriction activity but not the R.CviJI* activity of the native enzyme. Nine restriction and modification proteins which recognize a central GC or CG sequence share short regions of identity with R.CviJI amino acids 144-235, suggesting that this region is the recognition and/or catalytic domain.

Structural requirements for FokI-DNA interaction and oligodeoxyribonucleotide-instructed cleavage.

The FokI 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 between FokI and DNA, and the effect of Mg2+ on this interaction, we used FokI with various combinations of dsDNA, single-stranded (ss) DNA and oligodeoxyribonucleotides (oligos) containing a double-stranded hairpin carrying the FokI 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 using FokI 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 of FokI cohesive ends in the dsDNA, allowed FokI 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 with FokI (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 complex FokI and dsDNA was 12 minutes at 22 degrees 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.

GCN4 eukaryotic transcription factor/FokI endonuclease-mediated 'Achilles' heel cleavage': quantitative study of protein-DNA interaction.

Three proteins, yeast transcription regulatory protein GCN4, M.FokI DNA methyltransferase and R.FokI restriction endonuclease (ENase) were used to attain specific cleavage of DNA at the 18-20-bp GCN4 recognition site. This is a novel version of the 'Achilles' heel cleavage' (AC) technique [Koob et al., Science 241 (1988) 1084-1086]. Since the method employs a class-IIS ENase (R.FokI), which cleaves the DNA outside of its recognition sequence, it leaves the overlapping GCN4-binding intact. Thus, the same GCN4 site can be used in consecutive cleavage reactions. This novel GCN4-IIS-AC technique was applied to study the protein-DNA interaction. Quantitative analysis of the effect of temperature, reaction time, and GCN4 and M.FokI concentrations allowed determination of the GCN4-DNA complex half-life, which was found to be 7 h at 30 degrees C, 18 h at 22 degrees C and over 24 h at 10 degrees C. In addition, conditions for controlled, partial GCN4-IIS-AC digestion of DNA were determined, and applied to the physical mapping of large genomes.

Purification and characterization of the restriction endonuclease AvcI from Actinomyces cristalomycini.

The restriction endonuclease AvcI, an isoschizomer of Sau96I [Sussenbach et al., Nucleic Acids Res. 5 (1978) 1153-1163], was purified from Actinomyces cristalomycini. AvcI recognizes a 5-bp palindromic sequence, 5'-G decreases GNCC and cleaves it after the first G residue producing a 3-nucleotide 5'-overhang.

SacNI, an isoschizomer of BanII isolated from Streptomyces achromogenes recognizes the 5'-GRGCY/C sequence.

SacNI, an isoschizomer of the restriction endonuclease, BanII [Sugisaki et al., Nucleic Acids Res. 10 (1982) 5747-5752], has been isolated from Streptomyces achromogenes N-J-H. SacNI recognizes the palindromic sequence, 5'-GRGCY/C, and cleaves within the recognition sequence, generating a 3' protruding RGCY end (where R = A or G, and Y = C or G).

Cloning and applications of the two/three-base restriction endonuclease R.CviJI from IL-3A virus-infected Chlorella.

The gene (cviJIR) encoding the two/three-base R.CviJI eukaryotic restriction endonuclease (ENase) from IL-3A virus-infected Chlorella was cloned into Escherichia coli. A high frequency of DNA cleavage by R.CviJI required overexpression of the gene encoding the M.CviJI methyltransferase prior to cloning the gene for the ENase. Both genes were sequenced and their organization was determined to be in head-to-tail order. The open reading frame coding for R.CviJI can potentially translate a 41.4-kDa protein; however, in the E. coli host, a truncated version of the enzyme is produced (32.5 kDa). The recombinant ENase does not exhibit ATP-induced 'star' activity (R.CviJI cleaves at RGCY, while R.CviJI* also cleaves at RGCR and YGCY, but not at YGCR), as is characteristic for native R.CviJI. The very high frequency of DNA cleavage by R.CviJI* was exploited in the development of a quasi-random shotgun library method. R.CviJI*-generated oligodeoxyribonucleotides were applied to improve certain molecular biology applications, i.e., DNA labeling, detection, high-resolution restriction mapping, amplification and epitope mapping.

MmeI, a class-IIS restriction endonuclease: purification and characterization.

Two restriction endonucleases, MmeI and MmeII, from Methylophilus methylotrophus were purified to homogeneity. Both enzymes belong to the class-II restriction endonucleases (ENases) but exhibit very different enzymatic and physical properties. MmeII is a typical member of class-II ENases. It is a polymeric protein composed of 50-kDa subunits. In contrast to MmeII, MmeI is a monomeric protein of 101 kDa, cleaving a DNA molecule 20/18 nucleotides away from the asymmetric recognition sequence (5'-TCCRAC-3'); therefore, it is classified as a member of subclass-IIS. MmeI has an pI of 7.85 and is active in the pH range 6.5 to 10 with the optimum at 7 to 8. Increasing salt concentration creates an inhibitory effect on MmeI: 40 mM KCl decreases activity by 50%, 100 mM completely inhibits DNA cleavage. Tris.HCl (pH 7.5) at a concentration exceeding 20 mM inhibits MmeI activity. Mg2+ stimulates MmeI in the range of 0.2 to 35 mM, with the optimum between 0.5 and 10 mM.

Atypical DNA-binding properties of class-IIS restriction endonucleases: evidence for recognition of the cognate sequence by a FokI monomer.

The DNA-binding properties of the FokI restriction endonuclease were studied using the gel-mobility-shift assay. Specific recognition of the cognate sequence and cleavage of DNA are distinguishable functions and can be separated. FokI binds to its recognition site predominantly as a monomer. At high concentrations, FokI exhibits a cooperative recognition sequence-dependent aggregation. In 20 mM KCl/10 mM Tris.HCl buffer, the binding constant of FokI to its cognate site is equal 6.0-7.9 x 10(8)/mol and is lower than the values for most gene-regulatory proteins. FokI binding is 600-1500 times weaker to non-cognate double-stranded DNA than to the GGATG site, and 30,000 times weaker to single-stranded DNA or tRNA. The method of Bading [Nucleic Acids Res. 16 (1988) 5241-5248], used for determining the stoichiometry of protein bound to DNA by gel-mobility-shift assay, is extended.

Rapid shotgun cloning utilizing the two base recognition endonuclease CviJI.

A new approach has been developed for the rapid fragmentation and fractionation of DNA into a size suitable for shotgun cloning and sequencing. The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. Atypical reaction conditions which alter the specificity of this enzyme (CviJI**) yield a quasi-random distribution of DNA fragments from the small molecule pUC19 (2686 base pairs). To quantitatively evaluate the randomness of this fragmentation strategy, a CviJI** digest of pUC19 was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lacZ minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI** restricts PyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation. Advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 micrograms instead of 2-5 micrograms), fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed), and higher cloning efficiencies are obtained (CviJI** digested and column fractionated DNA transforms 3-16 times more efficiently than sonicated, end-repaired, and agarose fractionated DNA).

Purification and characterization of the FokI restriction endonuclease.

The restriction endonuclease FokI from Flavobacterium okeanokoites was purified to homogeneity. Based on gel filtration, sedimentation and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, the following properties of the enzyme were determined: FokI exists in one active monomeric form, and has an Mr of 64-65.4 x 10(3).FokI is a strongly basic protein with an isoelectric point of 9.4. The enzyme exhibits restriction activity in the pH range 5.0 to 10.5 (maximum level at pH 7.0-8.5) and its divalent cation requirement is satisfied not only by Mg2+, but also by Co2+, Mn2+, Ni2+, Cd2+, Zn2+ and Fe2+.

Construction of a DNA-polymerase I overproducing plasmid and isolation of the enzyme.

The polA gene of Escherichia coli coding for DNA polymerase I was cloned under the control of bacteriophage lambda promoter pL and gene N in a high copy number plasmid vector. The chromosomally located lambda cIts repressor gene kept the synthesis of the polA gene product at 28 degrees C at a low level. Raising the temperature to 43 degrees C resulted in inactivation of the repressor and overproduction of DNA polymerase I, which could easily be purified to homogeneity.