Esp1396I restriction-modification (RM) system recognizes an interrupted palindromic DNA sequence 5'-CCA(N)(5)TGG-3'. The Esp1396I RM system was found to reside on pEsp1396, a 5.6 kb plasmid naturally occurring in Enterobacter sp. strain RFL1396. The nucleotide sequence of the entire 5622 bp pEsp1396 plasmid was determined on both strands. Identified genes for DNA methyltransferase (esp1396IM) and restriction endonuclease (esp1396IR) are transcribed convergently. The restriction endonuclease gene is preceded by the small ORF (esp1396IC) that possesses a strong helix-turn-helix motif and resembles regulatory proteins found in PvuII, BamHI and few other RM systems. Gene regulation studies revealed that C.Esp1396I acts as both a repressor of methylase expression and an activator of regulatory protein and restriction endonuclease expression. Our data indicate that C protein from Esp1396I RM system activates the expression of the Enase gene, which is co-transcribed from the promoter of regulatory gene, by the mechanism of coupled translation.
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 ...
The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.
Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5′-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites. BpuJI is cleaved by chymotrypsin into an N-terminal domain (NTD), which lacks catalytic activity but binds specifically to the recognition sequence as a monomer, and a C-terminal domain (CTD), which forms a dimer with non-specific nuclease activity. Fold recognition approach reveals that the CTD of BpuJI is structurally related to archaeal Holliday junction resolvases (AHJR). We demonstrate that the isolated catalytic CTD of BpuJI possesses end-directed nuclease activity and preferentially cuts 3 nt from the 3′-terminus of blunt-ended DNA. The nuclease activity of the CTD is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the NTDs. This leads to a complicated pattern of specific DNA cleavage in the vicinity of the target site. Bioinformatics analysis identifies the AHJR-like domain in the putative Type III enzymes and functionally uncharacterized proteins.
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