Overproduction, purification and crystallization of TaqI restriction endonuclease

Under phoA promoter control, TaqI endonuclease was overproduced to 5% of Escherichia coli cellular proteins. This was achieved by fusing the endonuclease gene to the first four codons of the alkaline phosphatase signal sequence. For maximal overproduction (30% of cellular proteins), a putative 14-bp hairpin within the endonuclease coding sequence was replaced with degenerate codons. In addition, TaqI methylase was required to protect host DNA. The endonuclease was purified in sufficient amounts for crystallization.     

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+.     

Protein assembly and DNA looping by the FokI restriction endonuclease

The FokI restriction endonuclease recognizes an asymmetric DNA sequence and cuts both strands at fixed positions upstream of the site. The sequence is contacted by a single monomer of the protein, but the monomer has only one catalytic centre and forms a dimer to cut both strands. FokI is also known to cleave DNA with two copies of its site more rapidly than DNA with one copy. To discover how FokI acts at a single site and how it acts at two sites, its reactions were examined on a series of plasmids with either one recognition site or with two sites separated by varied distances, sometimes in the presence of a DNA-binding defective mutant of FokI. These experiments showed that, to cleave DNA with one site, the monomer bound to that site associates via a weak protein–protein interaction with a second monomer that remains detached from the recognition sequence. Nevertheless, the second monomer catalyses phosphodiester bond hydrolysis at the same rate as the DNA-bound monomer. On DNA with two sites, two monomers of FokI interact strongly, as a result of being tethered to the same molecule of DNA, and sequester the intervening DNA in a loop.     

Dynamics and consequences of DNA looping by the FokI restriction endonuclease

Genetic events often require proteins to be activated by interacting with two DNA sites, trapping the intervening DNA in a loop. While much is known about looping equilibria, only a few studies have examined DNA-looping dynamics experimentally. The restriction enzymes that cut DNA after interacting with two recognition sites, such as FokI, can be used to exemplify looping reactions. The reaction pathway for FokI on a supercoiled DNA with two sites was dissected by fast kinetics to reveal, in turn: the initial binding of a protein monomer to each site; the protein–protein association to form the dimer, trapping the loop; the subsequent phosphodiester hydrolysis step. The DNA motion that juxtaposes the sites ought on the basis of Brownian dynamics to take ~2 ms, but loop capture by FokI took 230 ms. Hence, DNA looping by FokI is rate limited by protein association rather than DNA dynamics. The FokI endonuclease also illustrated activation by looping: it cut looped DNA 400 times faster than unlooped DNA.     

Purification and crystallization of the EcoRV restriction endonuclease

The type II restriction endonuclease EcoRV purified from a genetically engineered, overproducing strain has been crystallized. Four crystal forms all obtained by precipitation with polyethylene glycol 4000 have been characterized. Two of these are suitable for high resolution structure analysis. Both are orthorhombic, have space group P2(1)2(1)2(1) and have similar unit cell dimensions of a = 58.2 A, b = 71.7 A, c = 130.6 A (form A) and a = 59.9 A, b = 74.5 A, c = 121.8 A (form B). They diffract to about 2A resolution and appear to have one dimer of 2 X 29,000 daltons in the asymmetric unit.     

Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

Many restriction endonucleases are dimers that act symmetrically at palindromic DNA sequences, with each active site cutting one strand. In contrast, FokI acts asymmetrically at a non-palindromic sequence, cutting ‘top’ and ‘bottom’ strands 9 and 13 nucleotides downstream of the site. FokI is a monomeric protein with one active site and a single monomer covers the entire recognition sequence. To cut both strands, the monomer at the site recruits a second monomer from solution, but it is not yet known which DNA strand is cut by the monomer bound to the site and which by the recruited monomer. In this work, mutants of FokI were used to show that the monomer bound to the site made the distal cut in the bottom strand, whilst the recruited monomer made in parallel the proximal cut in the top strand. Procedures were also established to direct FokI activity, either preferentially to the bottom strand or exclusively to the top strand. The latter extends the range of enzymes for nicking specified strands at specific sequences, and may facilitate further applications of FokI in gene targeting.     

Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance energy transfer

The FokI restriction endonuclease is a monomeric protein that recognizes an asymmetric sequence and cleaves both DNA strands at fixed loci downstream of the site. Its single active site is positioned initially near the recognition sequence, distant from its downstream target 13 nucleotides away. Moreover, to cut both strands, it has to recruit a second monomer to give an assembly with two active sites. Here, the individual steps in the FokI reaction pathway were examined by fluorescence resonance energy transfer (FRET). To monitor DNA binding and domain motion, a fluorescence donor was attached to the DNA, either downstream or upstream of the recognition site, and an acceptor placed on the catalytic domain of the protein. A FokI variant incapable of dimerization was also employed, to disentangle the signal due to domain motion from that due to protein association. Dimerization was monitored separately by using two samples of FokI labelled with donor and acceptor, respectively. The stopped-flow studies revealed a complete reaction pathway for FokI, both the sequence of events and the kinetics of each individual step.     

Overproduction of the EcoR V endonuclease and methylase.

Strains overproducing the EcoR V endonuclease and methylase have been obtained by inserting each of the two genes in expression vectors containing the lambda PL promoter. The methylase is overproduced to a level reaching 5-10% of the total cellular proteins, which represents a 50-100 fold increase. A 30 fold overproduction of endonuclease was achieved by randomly positioning the EndRV gene downstream of the lambda PL promoter. The situation in the endonuclease overproducing clone resembles that encountered in maxi-cells. The strains described here allowed a quick purification of both enzymes in sufficient amounts for crystallisation attempts.