Type IB enzymes

Type IB restriction endonucleases

Acknowledgement: Much of the material below is taken directly from the abstracts of published papers and as a consequence I have cited the relevant paper in each section. My thanks to all those workers who have contributed either knowingly or unknowingly to this page (KF).

The archetypical member of the Type IB family of Type I restriction-modification system is EcoAI (Suri et al., 1984). The recognition sequence of this enzyme is GAGN₆GTCA.

Domain structure of HsdS

Janscak et al. (1998) have subjected the HsdS subunit of the Type IB R-M enzyme EcoAI to circular permutation to investigate further the concept that the HsdS subunit fits the Kneale (1994). This was accomplished at the DNA level by direct linkage of codons for original termini and introduction of new termini elsewhere along the N-terminal and central conserved regions. By analysing the activity of mutant enzymes, two circularly permuted variants of HsdS that 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, suggesting a close proximity of the N and C termini in the native protein. The wild-type HsdS subunit was purified to homogeneity and shown to form a stable trimeric complex with HsdM, M₂S₁, which was fully active as a DNA methyltransferase. Gel electrophoretic mobility shift assays revealed that the HsdS protein alone was not able to form a specific complex with a 30-mer oligoduplex containing a single EcoAI recognition site. However, addition of stoichiometric amounts of HsdM to HsdS led to efficient specific DNA binding. Our data provide evidence for the circular organisation of domains of the HsdS subunit. In addition, they suggest a possible role of HsdM subunits in the formation of this structure.

DNA translocation/cleavage

Type I restriction enzymes bind to specific DNA sequences but subsequently translocate non-specific DNA past the complex in a reaction coupled to ATP hydrolysis and cleave DNA at any barrier that can halt the translocation process. The restriction subunit of these enzymes, HsdR, contains a cluster of seven amino acid sequence motifs typical of helicase superfamily II, that are believed to be relevant to the ATP-dependent DNA translocation. Alignment of all available HsdR sequences reveals an additional conserved region at the protein N-terminus with a consensus sequence reminiscent of the P-D...(D/E)-X-K catalytic motif of many Type I II restriction enzymes. To investigate the role of these conserved residues, Janscak et al. (1999) have produced mutants of the Type IB restriction enzyme EcoAI. They found that single alanine substitutions at Asp-61, Glu-76 and Lys-78 residues of the HsdR subunit abolished the enzyme's restriction activity but had no effect on its ATPase and DNA translocation activities, suggesting that these residues are part of the active site for DNA cleavage.

DNA supercoiling following translocation

Type I restriction enzymes cleave DNA at non-specific sites far from their recognition sequence as a consequence of ATP-dependent DNA translocation past the enzyme. During this reaction, the enzyme remains bound to the recognition sequence and translocates DNA towards itself simultaneously from both directions, generating DNA loops, which appear to be supercoiled when visualised by electron microscopy. To further investigate the mechanism of DNA translocation by Type I I restriction enzymes, Janscak and Bickle (2000) have probed the reaction intermediates with DNA topoisomerases. A DNA cleavage-deficient mutant of EcoAI, which has normal DNA translocation and ATPase activities, was used in these DNA supercoiling assays. In the presence of eubacterial DNA topoisomerase I, which specifically removes negative supercoils, the EcoAI mutant introduced positive supercoils into relaxed plasmid DNA substrate in a reaction dependent on ATP hydrolysis. The same DNA supercoiling activity followed by DNA cleavage was observed with the wild-type EcoAI endonuclease. Positive supercoils were not seen when eubacterial DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I, which removes both positive and negative supercoils. Furthermore, addition of eukaryotic DNA topoisomerase I to the product of the supercoiling reaction resulted in its rapid relaxation. These results are consistent with a model in which EcoAI translocation along the helical path of closed circular DNA duplex simultaneously generates positive supercoils ahead and negative supercoils behind the moving complex in the contracting and expanding DNA loops, respectively. In addition, we show that the highly positively supercoiled DNA generated by the EcoAI mutant is cleaved by EcoAI wild-type endonuclease much more slowly than relaxed DNA. This suggests that the topological changes in the DNA substrate associated with DNA translocation by Type I I restriction enzymes do not appear to be the trigger for DNA cleavage.

• Home • Up • Type IA enzymes • Type IB enzymes • Type IC enzymes • Type ID enzymes • New R-M •