Among all restriction endonucleases known to date, BfiI is unique in cleaving DNA in the absence of metal ions. BfiI represents a different evolutionary lineage of restriction enzymes, as shown by its crystal structure at 1.9-Å 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 BfiI exhibits a -barrel-like structure very similar to the effector DNA-binding domain of the Mg 2؉ -dependent restriction enzyme EcoRII and to the B3-like DNA-binding domain of plant transcription factors. BfiI 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 BfiI catalytic activity in the absence of a specific DNA sequence. A PSI-BLAST search identified a BfiI homologue in a Mesorhizobium sp. BNC1 bacteria strain, a plant symbiont isolated from an EDTA-rich environment.restriction endonuclease ͉ x-ray crystallography R estriction endonucleases (REases) protect bacteria by hydrolyzing invading viral or other foreign DNA. Type II REases perform their function by catalyzing the sequencespecific cleavage of double-stranded DNA molecules in the presence of Mg 2ϩ ions within or close to their recognition sites (1). Surprisingly, orthodox REases and bacteriophage -exonuclease that binds a free end of double-stranded DNA and processively degrades one strand in the 5Ј to 3Ј direction share a similar catalytic mechanism and a common structural ancestor (2). A -exonuclease-like domain has been identified in many Mg 2ϩ -dependent nucleases involved in DNA recombination and repair (3-5), suggesting that it has been remolded during evolution to perform different functions. To constrain cleavage at specific sites, REases had to develop effective means to control nucleolytic activity of -exonuclease-like catalytic domain.A variety of mechanisms have evolved to maintain REases in inactive configuration to avoid uncontrolled DNA cleavage and couple it to the recognition of specific nucleotide sequence. Orthodox REases, like EcoRI or BamHI, are homodimers that make largely symmetrical interactions with palindromic DNA sequences and contain two distinct sites each responsible for catalyzing cleavage in one DNA strand (1). In orthodox restriction enzymes, structural elements involved in sequence recognition are grafted on the conserved -exonuclease-like scaffold that makes a catalytic core (6). Structural analysis indicates that EcoRI amino acid residues involved in specific DNA binding are coupled to the catalytic residues through the intricate network of interactions (7). Therefore, any perturbation in the recognition site (for example, an incorrect base pair) would propagate via the network to the cleavage site and thus ...
EcoRII restriction endonuclease is specific for the 5′-CCWGG sequence (W stands for A or T); however, it shows no activity on a single recognition site. To activate cleavage it requires binding of an additional target site as an allosteric effector. EcoRII dimer consists of three structural units: a central catalytic core, made from two copies of the C-terminal domain (EcoRII-C), and two N-terminal effector DNA binding domains (EcoRII-N). Here, we report DNA-bound EcoRII-N and EcoRII-C structures, which show that EcoRII combines two radically different structural mechanisms to interact with the effector and substrate DNA. The catalytic EcoRII-C dimer flips out the central T:A base pair and makes symmetric interactions with the CC:GG half-sites. The EcoRII-N effector domain monomer binds to the target site asymmetrically in a single defined orientation which is determined by specific hydrogen bonding and van der Waals interactions with the central T:A pair in the major groove. The EcoRII-N mode of the target site recognition is shared by the large class of higher plant transcription factors of the B3 superfamily.
Methyltransferases (MTases) of procaryotes affect general cellular processes such as mismatch repair, regulation of transcription, replication, and transposition, and in some cases may be essential for viability. As components of restriction-modification systems, they contribute to bacterial genetic diversity. The genome of Helicobacter pylori strain 26695 contains 25 open reading frames encoding putative DNA MTases. To assess which MTase genes are active, strain 26695 genomic DNA was tested for cleavage by 147 restriction endonucleases; 24 were found that did not cleave this DNA. The specificities of 11 expressed MTases and the genes encoding them were identified from this restriction data, combined with the known sensitivities of restriction endonucleases to specific DNA modification, homology searches, gene cloning and genomic mapping of the methylated bases m 4 C, m 5 C, and m 6 A.The bacterium Helicobacter pylori chronically infects the upper gastrointestinal tract of the majority of people worldwide and is major cause of chronic gastritis and peptic ulcer disease and an early risk factor for gastric cancer (39). H. pylori strains are diverse genetically (1,2,4,16,21,44), and certain strain differences may contribute to the diversity of clinical outcome of infection (13,15,47). Analyses of genome sequences of H. pylori strains 26695 (46) and J99 (3) revealed that each strain contained more than two dozen genes likely to encode DNA methyltransferases (MTases), far more than have been detected in other bacterial genomes to date (20). These MTase genes comprise a large fraction of all genes that were specific to one or the other strain. Bacterial MTases exist either as components of restriction-modification (R-M) systems or as separate enzymes. The former are strain specific, and their main function is to protect host DNA from damage by a cognate restriction enzyme (RE). Separate MTases, which tend to be present in all strains of a species (species-specific MTases), are involved in general cellular processes such as regulation of transcription of specific genes (5, 48), DNA replication (32), mismatch repair (33), or DNA transposition (40). Certain of them can be essential for viability (43) or contribute to specificity in bacterium-host interactions or virulence in pathogens (8,17).Most MTase genes found in the genomes of H. pylori strains 26695 and J99 probably belong to R-M systems. That certain R-M systems might also affect bacterium-host interactions was suggested by induction of transcription of iceA1, a homolog of RE NlaIII, by H. pylori-host cell contact (36). Although iceA1 does not encode a functional protein in most H. pylori strains (14), it is adjacent to a generally active MTase gene (hpyIM) specific for sites recognized by NlaIII. Thus, the hpyI-iceA1 pair may represent a vestige of an R-M system that has evolved some special function relevant to host-pathogen interactions (14). By extrapolation, the same might be true for the MTases of other putative R-M systems in H. pylori. The identification ...
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