Type I Restriction and Modification Systems

The genes of Type I R-M Systems

Type I Restriction-Modification enzymes are composed of three subunits, encoded by the three host specificity determinant (hsd) genes; the hsdR gene is absolutely required for restriction activity; the hsdS gene is responsible for DNA specificity and together with hsdM is the minimal genetic requirement for methylation activity. However, the holoenzyme can also behave as a methylase, the activity of the enzyme is dependent upon the methylation status of the DNA substrate. Therefore, the enzyme must control the opposing functions of restriction and modification.

hsdR - restriction gene, phenotype of mutations = r^-m⁺.

hsdM - modification gene, phenotype of mutations = r^-m^-.

hsdS - specificity gene, phenotype of mutations = r^-m^-.

The domain structure of HsdS

The DNA-binding subunit of a Type I R-M system consists of two target recognition domains separated by conserved domains, which are highly conserved between family members.

For the EcoR124I R-M system the recognition sequence (s_R124) is 5'-GAAnnnnnnRTCG-3'. TRD1 is responsible for recognition of the 5'-end of the recognition sequence (GAA), while TRD2 recognises the 3'-end (RTCG).

The conserved regions contain two highly conserved repeats (shown by the arrows) that were originally described by Argos (1983) as the regions of the protein likely to recognise the target DNA sequence. However, this has now been shown not to be the case (e.g. Gubler, 1992).

Careful analysis of the arrangement of these repeated units in all Type I HsdS subunits indicates that all the subunits consist of a similar set of sub-repeats and this has led to a prediction of a circular structure for the HsdS subunit (Kneale 1994).    To test this model, Janscak & Bickle (1998) circularly permuted the HsdS subunit of the Type IB R-M enzyme EcoAI, at the DNA level, by direct linkage of codons for the original termini and introduction of new termini elsewhere along the N-terminal and central conserved regions. By analysing the activity of these mutant enzymes, two circularly permuted variants of HsdS, which had termini located at equivalent positions in the N-terminal and central repeats, respectively, were found to fold into a functional DNA recognition subunit with wild-type specificity.  This result suggests that the N- and C-termini of the native protein are in close proximity.

Recently, two crystal structures have been solved for HsdS proteins from the uncharacterised Type I R-M systems MjaXIP (Kim et al., 2005) and MgeORF438 (Calisto et al., 2005) respectively.  While these HsdS subunits are not known to be components of functional restriction enzymes, and their DNA specificity is unknown, the structure has confirmed the circular nature of the HsdS subunit and the close proximity of the N- and C-termini as indicated by Janscak & Bickle.

The central conserved region has been subjected to intensive mutagenesis using deletion mutagenesis, site-directed mutagenesis and PCR-misincorporation mutagenesis. This has allowed us to show that this region is critical for protein-protein interactions with the other subunits of the endonuclease (Abadjieva et al., 1994).  TRD1 and the central conserved region are sufficient to assemble an active enzyme (Abadjieva et al., 1993; Macwilliams & Bickle, 1996). This reflects the circular structure of the HsdS subunit and the deletion mutants assemble through dimerisation of a M₁S_½ subunit. Since the recognition sequence of this deletion mutant is GAAnnnnnnnTTC, and TRD1 recognises the GAA component of the s_R124 sequence, it is apparent that each half subunit recognises the opposite strand on the DNA strand.

The Type IC DNA methyltransferase M.EcoR124I recognises the nonpalindromic DNA sequence GAAn₆RTCG. Taylor et al. (1994) have used small angle X-ray scattering to investigate the solution structure of the methyltransferase and of complexes of the enzyme with unmethylated and hemimethylated 30 bp DNA duplexes containing the specific recognition sequence. A major change in the quaternary structure of the enzyme was observed following DNA binding, based on a decrease in the radius of gyration and a reduction in the maximum dimension of the enzyme. The structural transition observed is independent of the methylation state of the DNA. CD shows that there is no change in the secondary structure of the protein subunits when DNA is bound. In contrast, there is a large increase in the CD signal arising from the DNA, suggesting considerable structural distortion which may allow access to the bases targeted for methylation. We propose that DNA binding induces a large rotation of the two HsdM subunits towards the DNA, mediated by hinge bending domains in the specificity subunit HsdS.

van Noort et al. (2004) have shown that the large structural distortion in the DNA is due to DNA bending and that the MTase induces a DNA bend of 49º.  This bend is comparable to that observed with EcoKI, which is also the angle of the 'mimic' protein OCR (Walkinshaw et al 2002), which acts an antirestriction protein (Atanasiu et al 2002), and is presumed compete for the DNA target site within the enzyme.

DNA specificity

Type I restriction-modification enzymes recognise bipartite sequences, which consist of a 5' specific region of 3-4 bp and a 3' specific region of, usually, 4 bp, separated by a non-specific spacer of 6-8 bp (e.g. EcoR124I recognises GAAn₆RTCG - where R = purine and n = any nucleotide). DNA cleavage by Type I R-M enzymes occurs at non-specific sites far from their recognition sequence, which is mediated by ATP-dependent DNA translocation past the enzyme (Yuan et al., 1980; Studier & Banyopadhyay 1888; Dryden et al., 1997; Szczelkun et al., 1997).

Known recognition sequences for Type I R-M enzymes:

Last modified on 24 July 2010
© University of Portsmouth
Author Dr Keith Firman.