ABSTRACT
BACKGROUND: Restriction endonucleases belong to prokaryotic restriction-modification systems, that protect host cells from invading DNA. Type II restriction endonucleases recognize short 4-8 bp sequences in the target DNA and cut both DNA strands producing double strand breaks. Type II restriction endonuclease Kpn2I cleaves 5'-T/CCGGA DNA sequence ("/" marks the cleavage position). Analysis of protein sequences suggested that Kpn2I belongs to the CCGG-family, which contains ten enzymes that recognize diverse nucleotides outside the conserved 5'-CCGG core and share similar motifs for the 5'-CCGG recognition and cleavage. METHODS: We solved a crystal structure of Kpn2I in a DNA-free form at 2.88 Å resolution. From the crystal structure we predicted active center and DNA recognition residues and tested them by mutational analysis. We estimated oligomeric state of Kpn2I by SEC-MALS and performed plasmid DNA cleavage assay to elucidate DNA cleavage mechanism. RESULTS: Structure comparison confirmed that Kpn2I shares a conserved active site and structural determinants for the 5'-CCGG tetranucleotide recognition with other restriction endonucleases of the CCGG-family. Guided by structural similarity between Kpn2I and the CCGG-family restriction endonucleases PfoI and AgeI, Kpn2I residues involved in the outer base pair recognition were proposed. CONCLUSIONS: Kpn2I is an orthodox Type IIP restriction endonuclease, which acts as a dimer. Kpn2I shares structural similarity to the CCGG-family restriction endonucleases PfoI, AgeI and PspGI. GENERAL SIGNIFICANCE: The Kpn2I structure concluded the studies of the CCGG-family, covering detailed structural and biochemical characterization of eleven restriction enzymes and their complexes with DNA.
Subject(s)
Bacterial Proteins/chemistry , Crystallography, X-Ray/methods , Endodeoxyribonucleases/chemistry , Amino Acid Sequence , Bacterial Proteins/metabolism , Binding Sites , Catalytic Domain , Endodeoxyribonucleases/metabolism , Models, Molecular , Molecular Structure , Protein Binding , Sequence Homology, Amino AcidABSTRACT
A bacterial strain 2H isolated from soil and identified as Thermoactinomyces vulgaris produce a potent Type II restriction endonuclease activity that has been extracted by a PEG/dextran aqueous two-phase system. Optimal temperature for the restriction endonuclease activity was 55-65°C. Specific DNA cleavage was obtained at pH range 7-10 and 10-20mM MgCl2. Restriction cleavage analysis followed by sequencing confirms GG^CC as the recognition sequence. This enzyme, named Tvu2HI, is a thermostable isoschizomer of the mesophilic prototype restriction endonuclease HaeIII. Sequencing of the complete Thermoactinomyces vulgaris 2H genome revealed the presence of two adjacent ORFs coding for the restriction endonuclease Tvu2HI and the corresponding methyltransferase; an ORF coding for a putative Vsr nicking enzyme was found close to those coding for the Tvu2HI restriction-modification system. Phylogenetic analysis based on sequence alignment suggests a common origin of Tvu2HI R-M system with HaeIII-like R-M systems. This is the first investigation dealing with a Type II restriction endonuclease identified in a natural isolate of the genus Thermoactinomyces.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/genetics , Deoxyribonucleases, Type II Site-Specific/metabolism , Thermoactinomyces/classification , Whole Genome Sequencing/methods , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Deoxyribonucleases, Type II Site-Specific/chemistry , Enzyme Stability , Evolution, Molecular , Hydrogen-Ion Concentration , Open Reading Frames , Phylogeny , Soil Microbiology , Substrate Specificity , Thermoactinomyces/enzymology , Thermoactinomyces/genetics , Thermoactinomyces/isolation & purification , ThermodynamicsABSTRACT
A moderately thermophilic Gram-negative bacterium isolated from the Polok hot spring, Sikkim, India, was identified as a strain (PL17) of Tepidimonas fonticaldi by 16S rDNA sequencing. T. fonticaldi PL17 produces a Type IIP restriction endonuclease; named TfoI. Restriction mapping, run-off sequencing of TfoI-digests of dsDNA fragments, and end compatibility of TfoI with NdeI confirmed that the enzyme recognizes and cleaves the sequence 5'-T^TAA-3', and is thus an isoschizomer of MseI. The TfoI restriction-modification genes in the T. fonticaldi PL17 genome were identified, and the annotated TfoI protein encodes a protein of 181 amino acid residues that shares 47.2% sequence identity with MseI. The native enzyme was purified using a four-column chromatography protocol, and its functional homogeneity was confirmed by standard quality control tests. The ESI-MS measured molecular weight of purified TfoI (20.696 kDa) is in agreement with that of the calculated monomeric molecular weight of the predicted TfoI protein sequence (20.694 kDa). TfoI exhibits optimal activity in the temperature range of 55-70 °C with Mg+2 or Co+2 as cofactor. Similar to its isoschizomers, TfoI can be used as the frequent cutter for genome analysis.
Subject(s)
Bacterial Proteins/metabolism , Betaproteobacteria/enzymology , Deoxyribonucleases, Type II Site-Specific/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Betaproteobacteria/genetics , Deoxyribonucleases, Type II Site-Specific/chemistry , Deoxyribonucleases, Type II Site-Specific/genetics , Enzyme Stability , Isoenzymes , Substrate SpecificityABSTRACT
A strain of Bacillus pumilus was isolated and identified from water samples collected from a small affluent of the Amazon River. Type II restriction endonuclease activity was detected in these bacteria. The enzyme was purified and the molecular weight of the native protein estimated by gel filtration and SDS-PAGE. The optimum pH, temperature and salt requirements were determined. Quality control assays showed the complete absence of "nonspecific nucleases." Restriction cleavage analysis and DNA sequencing of restriction fragments allowed the unequivocal demonstration of 5´GAG FONT FACE=Symbol>¯ /FONT>CTC3´ as the recognition sequence. This enzyme was named BpuAmI and is apparently a neoschizomer of the prototype restriction endonuclease SacI. This is the first report of an isoschizomer and/or neoschizomer of the prototype SacI identified in the genus Bacillus.
Uma linhagem de Bacillus pumilus foi isolada e identificada de amostras de águas coletadas em um pequeno Igarapé do Rio Amazonas. Foi detectada atividade de restrição do tipo II nesta bactéria. A enzima foi purificada e o peso molecular da proteína nativa foi estimado por gel filtração e por eletroforese em gel de poliacrilamida. Foram determinados, o pH e temperatura ótimos e as necessidades de sais. Os ensaios do controle de qualidade mostraram uma ausência completa de "nucleases não específicas". As analises das clivagens e o seqüenciamento do DNA dos fragmentos de restrição permitiram uma demonstração inequívoca de que 5´GAG FONT FACE=Symbol>¯ /FONT>CTC 3´ é a seqüência de reconhecimento da enzima. Esta enzima foi denominada de BpuAmI e aparentemente é um neoesquisômero da enzima protótipo SacI. Este é o primeiro relato de um isoesquisômero e/ou neoesquisômero da enzima protótipo SacI identificada no gênero Bacillus.
