Crystallization and preliminary X-ray diffraction analysis of MspI restriction endonuclease in complex with its cognate DNA.

The MspI restriction endonuclease is a type II restriction enzyme. Unlike all other restriction enzymes with known structures, MspI recognizes the palindromic tetranucleotide sequence 5'-C/CGG and cleaves it as indicated by the '/' to produce DNA products with 5' two-base overhangs. Owing to the nature of its cleavage pattern, it is likely that MspI would represent a new structural class of restriction endonucleases. Crystals of the dimeric MspI restriction enzyme bound to a duplex DNA molecule containing the specific recognition sequence have been obtained by vapor-diffusion techniques in the presence of polyethylene glycol as precipitant. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 50.2, b = 131.6, c = 59.3 A, beta = 109.7 degrees. The crystals contain one dimeric complex in the asymmetric unit. A complete native data set has been collected to a resolution of 2.05 A by cryo-crystallographic methods, with an R(merge) of 4.0%.

Crystallization and preliminary diffraction analysis of a hyperthermostable DNA polymerase from a Thermococcus archaeon.

The hyperthermostable DNA polymerase from a marine Thermococcus archaeon has been crystallized in space group P212121, with unit-cell dimensions a = 94.8, b = 98.2, c = 112.2 A with one molecule per asymmetric unit. Conditions for data collection at 98 K have been identified, and a complete data set was collected to 2.2 A resolution. Strategies employed here may facilitate crystallization of other hyperthermostable proteins. The structure of this enzyme will provide the first structural data on the archaeal and hyperthermostable classes of DNA polymerases. Sequence homology to human polymerase alpha (polymerase B family) may make it a model for studying eukaryotic and viral polymerases and for the development of anti-cancer and anti-viral therapeutics.

Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element.

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.

Cloning, expression and sequence analysis of the SphI restriction-modification system.

SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5'-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3' end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.

Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on Thermococcus sp. 9 degrees N-7 and mutations affecting 3'-5' exonuclease activity.

Five extremely thermophilic Archaea from hydrothermal vents were isolated, and their DNA polymerases were cloned and expressed in Escherichia coli. Protein splicing elements (inteins) are present in many archaeal DNA polymerases, but only the DNA polymerase from strain GB-C contained an intein. Of the five cloned DNA polymerases, the Thermococcus sp. 9 degrees N-7 DNA polymerase was chosen for biochemical characterization. Thermococcus sp. 9 degrees N-7 DNA polymerase exhibited temperature-sensitive strand displacement activity and apparent Km values for DNA and dNTP similar to those of Thermococcus litoralis DNA polymerase. Six substitutions in the 3'-5' exonuclease motif I were constructed in an attempt to reduce the 3'-5' exonuclease activity of Thermococcus sp. 9 degrees N-7 DNA polymerase. Five mutants resulted in no detectable 3'-5' exonuclease activity, while one mutant (Glul43Asp) had <1% of wild-type activity.

Cloning and expression of the NaeI restriction endonuclease-encoding gene and sequence analysis of the NaeI restriction-modification system.

NaeI, a type-II restriction-modification (R-M) system from the bacterium Nocardia aerocolonigenes, recognizes the sequence 5'-GCCGGC. The NaeI DNA methyltransferase (MTase)-encoding gene, naeIM, had been cloned previously in Escherichia coli [Van Cott and Wilson, Gene 74 (1988) 55-59]. However, none of these clones expressed detectable levels of the restriction endonuclease (ENase). The absence of the intact ENase-encoding gene (naeIR) within the isolated MTase clones was confirmed by recloning the MTase clones into Streptomyces lividans. The complete NaeI system was finally cloned using E. coli AP1-200 [Piekarowicz et al., Nucleic Acids Res. 19 (1991) 1831-1835] and less stringent MTase-selection conditions. The naeIR gene was expressed first by cloning into S. lividans, and later by cloning under control of a regulated promoter in an E. coli strain preprotected by the heterologous MspI MTase (M.MspI). The DNA sequence of the NaeI R-M system has been determined, analyzed and compared to previously sequenced R-M systems.

Characterization of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis. Vent DNA polymerase, steady state kinetics, thermal stability, processivity, strand displacement, and exonuclease activities.

We have isolated, cloned, and characterized a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis, the Tli DNA polymerase (also referred to as Vent DNA polymerase). The enzyme is extremely thermostable, having a half-life of 8 h at 95 degrees C and about 2 h at 100 degrees C. Pseudo-first-order kinetics at 70 degrees C reveal an extremely low Km for a primed M13mp18 substrate (0.1 nM), coupled with a relatively high Km for dNTPs (50 microM). Accompanying extension rates are on the order of 1000 nucleotides/min. Synthesis by the polymerase is largely distributive, adding an average of 7 nucleotides/initiation event. This distributive synthesis can generate products of at least 10,000 bases. Tli DNA polymerase contains a 3'-->5' exonuclease activity that enhances the fidelity of replication by the enzyme (Mattila, P., Korpela, J., Tenkanen, T. and Pitkanen, K. (1991) Nucleic Acids Res. 19, 4967-4973). A 2-amino acid substitution within the conserved exonuclease domain abolishes both double and single strand-dependent exonuclease activity, without altering kinetic parameters for polymerization on a primed single-stranded template. Strand displacement activity by the mutated and unmutated forms increases with increasing temperature and is enhanced in the exonuclease-deficient form of the enzyme.

Intervening sequences in an Archaea DNA polymerase gene.

The DNA polymerase gene from the Archaea Thermococcus litoralis has been cloned and expressed in Escherichia coli. It is split by two intervening sequences (IVSs) that form one continuous open reading frame with the three polymerase exons. To our knowledge, neither IVS is similar to previously described introns. However, the deduced amino acid sequences of both IVSs are similar to open reading frames present in mobile group I introns. The second IVS (IVS2) encodes an endonuclease, I-Tli I, that cleaves at the exon 2-exon 3 junction after IVS2 has been deleted. IVS2 self-splices in E. coli to yield active polymerase, but processing is abolished if the IVS2 reading frame is disrupted. Silent changes in the DNA sequence at the exon 2-IVS2 junction that maintain the original protein sequence do not inhibit splicing. These data suggest that protein rather than mRNA splicing may be responsible for production of the mature polymerase.