Macroevolution by transposition: drastic modification of DNA recognition by a type I restriction enzyme following Tn5 transposition.

Abstract

We have characterized a novel mutant of EcoDXXI, a type IC DNA restriction and modification (R‐M) system, in which the specificity has been altered due to a Tn5 insertion into the middle of hsdS, the gene which encodes the polypeptide that confers DNA sequence specificity to both the restriction and the modification reactions. Like other type I enzymes, the wild type EcoDXXI recognizes a sequence composed of two asymmetrical half sites separated by a spacer region: TCA(N7)RTTC. Purification of the EcoDXXI mutant methylase and subsequent in vitro DNA methylation assays identified the mutant recognition sequence as an interrupted palindrome, TCA(N8)TGA, in which the 5′ half site of the wild type site is repeated in inverse orientation. The additional base pair in the non‐specific spacer of the mutant recognition sequence maintains the proper spacing between the two methylatable adenine groups. Sequencing of both the wild type and mutant EcoDXXI hsdS genes showed that the Tn5 insertion occurred at nucleotide 673 of the 1221 bp gene. This effectively deletes the entire carboxyl‐terminal DNA binding domain which recognizes the 3′ half of the EcoDXXI binding site. The truncated hsdS gene still encodes both the amino‐terminal DNA binding domain and the conserved repeated sequence that defines the length of the recognition site spacer region. We propose that the EcoDXXI mutant methylase utilizes two truncated hsdS subunits to recognize its binding site. The implications of this finding in terms of subunit interactions and the malleability of the type I R‐M systems will be discussed.

Type I Restriction Systems: Sophisticated Molecular Machines (a Legacy of Bertani and Weigle)

“…The present model (Fig. 2c) and that of Willcock et al (191) indicate a methyltransferase structure with twofold rotational symmetry in which inversely oriented TRDs will each make contact with one HsdM subunit (113). This symmetrical configuration of the domains within HsdS is supported by the following observations.…”

“…This symmetrical configuration of the domains within HsdS is supported by the following observations. (i) Truncated forms of the HsdS subunit of either EcoDXXI or EcoR124I, in which the carboxy half of the polypeptide is missing but the central conserved sequence is retained, associate to form an active enzyme that recognizes a bipartite target sequence that is a palindromic version of the trinucleotide specified by the amino-terminal TRD (1,113). Hence, EcoDXXI recognizes TCA(N 7 )RTTC, while the derivative with a truncated HsdS polypeptide recognises TCA(N 8 )TGA (Fig.…”

Immigration control of DNA in bacteria: self versus non-self

“…Early type I R-M systems with the subunit composition R # M # S # are likely to have recognized hyphenated symmetrical sequences, dictated by the symmetrical arrangement of two specificity subunits. Enzymes of this sort have been generated by deletions that truncate a specificity gene leading to an active enzyme comprising two symmetrically arranged truncated subunits (Abadjieva et al, 1993 ;Meister et al, 1993). Diversification of TRDs has led to the recognition of a variety of target sequences comprising 3-5 bp but always sequences within which an adenine residue is the substrate for methylation.…”

Families of restriction enzymes: an analysis prompted by molecular and genetic data for type ID restriction and modification systems

“…The variability in the IC family is dictated by whether a tetrapeptide sequence (TAEL) within the central conserved region is present in duplicate or in triplicate, the additional four amino acids increasing the separation of the target adenines by 1 bp (39). The importance of the correct spacing between the adenine residues is emphasised by the target sequences for EcoR124I∆ (40) and EcoDXXI∆ (41). In these systems, where the 3′ half of hsdS is lost and the two truncated HsdS subunits associate symmetrically, an additional base pair within the spacer sequence maintains the distance between the target adenines ( Table 3).…”