Two DNA recognition domains of the specificity polypeptides of a family of type I restriction enzymes.

Abstract

The hsd genes of Salmonella typhimurium and Salmonella potsdam encode related type I restriction and modification systems designated SB and SP, respectively; the polypeptide encoded by the hsdS gene dictates the DNA sequence recognized. The hsdS genes of the SB and SP systems have a conserved sequence of around 100 base pairs flanked by two nonhomologous (variable) regions of around 500 base pairs. Recombination between the hsdS genes of SB and SP generated a system (SQ) with a different recognition specificity.We have localized the position of the crossover in the central conserved region by analysis of nucleotide sequences. Concomitant with the generation of a new combination of flanking variable regions is the recombination of minor differences in the central conserved region. A polypeptide domain encoded on the 5' side of the crossover dictates recognition of the trinucleotide component of the target sequence, and a second domain, encoded on the 3' side of the crossover, similarly governs recognition of the tetra-or penta-nucleotide component. Our analysis implicates at least parts of the variable regions in the determination of the specificity of interaction between protein and DNA. Furthermore, the trinucleotide components of the recognition sequences of S. typhimurium and Escherichia coli K-12 are identical, and the 5' segments of their hsdS genes are strikingly homologous rather than variable.Some strains of Escherichia coli and Salmonella spp have type I restriction systems that are related to that of E. coli K-12 (K system). The concept of a family of enzymes, members of which carry out essentially the same reactions following the recognition of different DNA sequences, poses questions concerning both the specific interaction of the enzyme with its target sequence and the evolution ofdifferent specificities of interaction. Type I restriction endonucleases are encoded by three chromosomal genes: hsdR, hsdM, and hsdS. Genetic experiments show that the corresponding polypeptides (R, M, and S) can be interchanged between related systems and identify the S subunit as the determinant of the specificity of recognition (1-3). The various S polypeptides interact with related polypeptides to produce modification enzymes and with M and R subunits in the formation of restriction endonucleases. Related S subunits must retain a basic similarity for their interactions with other M and R polypeptides but have diverged to enable recognition of different nucleotide sequences.The hsdS genes of related E. coli systems (K, B, and D) have only localized DNA sequence homology (4). The genes vary in length from 1335 to 1425 base pairs (bp), and the regions of homology are -100 bp in the middle and 250 bp at the 3' end. Although these two regions are highly conserved, the remainder of each hsdS gene shares little or no homology with either of the other two.The K family includes the SB system found in Salmonella typhimurium and the SP system of Salmonella potsdam. When the SP hsd genes were transferred by phage P1 trans...

Characterization of mutations of the bacteriophage P1 mod gene encoding the recognition subunit of the EcoP1 restriction and modification system

“…These are considerably more complicated than the type III systems described here (for reviews, see references 6, 7, and 38) but have one property in common with the type III systems: a single polypeptide, the product of the hsdS gene, provides DNA sequence recognition in both restriction and modification. For the family of type I enzymes that includes EcoK, the sequence of the hsdS genes of several family members has been determined (10)(11)(12). The results are amazingly similar to those found with the type III mod genes in that the hsdS genes are also a mosaic of conserved and nonconserved regions.…”

EcoR124I: from Plasmid-Encoded Restriction-Modification System to Nanodevice

“…Further work by Gubler and Bickle not only confirmed this hypothesis but also showed the upper and lower limits of such expansion (32). That work supported the growing evidence that type I R-M systems were highly adaptable with respect to their abilities to generate novel DNA specificities, especially through recombination events including domain swapping (4,24,25).…”

“…The difference between the two specificities was unexpectedly simple: EcoR124I was shown to recognize GAAn 6 RTCG, while EcoR124/3 was shown to recognize the same specific bases but with the inclusion of an extra nonspecific base in the spacer region of the recognition sequence (GAAn 7 RTCG). This was an exciting observation, as it supported the two-domain model for HsdS (originally proposed by Argos [4] and later extended in studies by Murray and others [24,25]) in which a spacer region separates two target recognition domains (TRDs) that bind the specific nucleotides in the recognition sequence. It seemed likely that this spacer was extended in EcoR124/3.…”

Molecular characterization of the lactococcal plasmid pCIS3: natural stacking of specificity subunits of a type I restriction/modification system in a single lactococcal strain The GenBank accession numbers for the sequences reported in this paper are AF153410–AF153414.

“…It was shown that the first variable domain specifies the 5h DNA recognition sequence whereas the second specifies the 3h DNA recognition sequence, with a spacing of 6-8 nt between both (FullerPace & Murray, 1986). As a consequence of their structure, variable domains can be swapped between the DNA regions encoding HsdS subunits via crossing-over events, resulting in novel hsdS genes with altered specificities (Fuller-Pace & Murray, 1986). Type I R\M systems have recently been shown to be widespread in lactococci and they could well be the most abundant R\M systems present in this group of bacteria (Schouler et al, 1998a, b).…”