A deletion mutant of the type IC restriction endonuclease EcoR124l expressing a novel DNA specificity

Abstract

We have developed a complementation assay which allows us to distinguish between mutations affecting subunit assembly and mutations affecting DNA binding in the DNA recognition subunit (HsdS) of the multimeric restriction endonuclease EcoR1241. A number of random point mutations were constructed to test the validity of this assay. Two of the mutants produced were found to be truncated polypeptides that were still capable of complementation with the EcoR1241 Hsd subunits to give an active restriction enzyme of novel DNA specificity. The N-terminal variable domain (responsible for recognition of GAA from the EcoR1241 recognition sequence GAAnnnnnnRTCG) and the spacer region (central conserved region) is intact in both of these mutants. One of these mutant genes (hsdS(delta 50) has been cloned as an active Mtase. Purification of the Mtase proved to be difficult because the complex is weak. However, Mtase activity was obtained from a soluble cell extract, and this allowed us to determine the DNA recognition sequence of the Mtase to be GAAnnnnnnnTTC. This recognition sequence is an inverted repeat of 5'-end of the EcoR1241 recognition sequence. This suggests that the mutant Mtase is assembled from two inverted HsdS(D50) subunits, possibly held together by the HsdM subunits.

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

“…3a) imply that two "half" specificity subunits (HsdS*) within one TRD can substitute for a single wild-type HsdS with two TRDs (1,113). This is consistent with a complex of R 2 M 2 S* 2 rather than the normal R 2 M 2 S 1 .…”

“…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.…”

“…Two truncated polypeptides can substitute for one normal HsdS subunit if the subunits retain their ability to interact with HsdM. Active complexes, i.e., those in which HsdS retains the ability to bind HsdM, are recognized by their new specificities (1,109,113). Analyses of truncated polypeptides implicate the three conserved regions of HsdS (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.…”

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

“…In hindsight, perhaps one interesting comment from Argos was the idea of a "pseudodimer," based on the idea of repeating domains. Described later in this review is the existence of such a pseudodimer consisting of a repeating half-HsdS subunit (1,54). At the same time as Argos published his paper on repeating domains within the HsdS subunit of type I R-M enzymes, Fuller-Pace et al (23) described a rearrangement of DNA specificity based on recombination between conserved regions within hsdS that "swapped" DNA specificities by swapping domains within the HsdS subunit.…”