1. David Dryden School of Chemistry University of Edinburgh Bacterial DNA restriction and modification systems: structure, function and evolution.

2. Current group: Gareth Roberts, John White, Laurie Cooper, Ed Bower, Matt Tilling Collaborators: University of Cambridge: Robert Henderson, Mike Edwardson University of Edinburgh: Noreen Murray, Garry Blakely, Malcolm Walkinshaw, Wilson Poon, Anita Jones St. George's, University of London: Patrick Houston, Jodi Lindsay University of Leeds: Chris Kennaway, John Trinick University of Portsmouth: James Taylor, Geoff Kneale University of St. Andrews: Jim Naismith Bacterial DNA restriction and modification systems: structure, function and evolution.

3. 1. Restriction and modification (RM) systems and their inhibition by antirestriction systems. 2. Type I RM enzymes: structure, function and evolution. 3. Some speculation on the antiquity of RM systems and their influence on evolution.

4. RM systems control Horizontal Gene Transfer. Tock and Dryden, Curr. Op. Microbiol. (2005) 8, 466.

5. The major classes of RM systems. There are 11 subtypes of Type II systems! Also Type III and Type IV systems. 1190Archaea: 259 No RM 4651 With RM Bacteria: REBASE Genomes (4910 Bacteria, 191 Archaea)

6. +Mg 2+ ATP SAM The tools of molecular biology are the simplest Type II restriction enzymes. Nature has evolved systems of far greater complexity such as the Type I systems. Type I RM enzymes comprise 5 subunits 1x HsdS binds DNA target 2x HsdM binds SAM, methylates DNA 2xHsdR hydrolyses ATP, translocates and cuts DNA Roberts et al. Nucleic Acids Res. (2003) 31, 1805 Dryden Nature Struct. Mol. Biol. (2005) 11, 804. The major classes of RM systems are Type I and Type II systems.

7. Antirestriction methods: 1. Avoid having target sites 2. Modified DNA bases 3. Co-injection of antirestriction proteins 4. Consumption of cofactors for R/M systems 5. Enhance host modification activity 6. Proteolysis of RM enzymes 7. Encode specialised antirestriction proteins e.g. ArdA and ArdB on conjugative transposons and plasmids and Ocr on phage T7. Plus many others as yet poorly characterised. Bickle and Kruger, Microbiol. Rev. (1993) 57, 434. Tock and Dryden, Curr. Op. Microbiol. (2005) 8, 466. Dryden, Trends Biotech. (2006) 24, 378. Walkinshaw et al. Mol. Cell (2002) 9, 187. McMahon et al., (2009) Nucl. Acids Res. 37, 4887. Serfiotis-Mitsa et al., (2009) Nucl. Acids Res. 38,1723. Antirestriction systems such as antirestriction proteins control RM systems. They co-evolve to assist Horizontal Gene Transfer. Ocr from phage T7

8. Antirestriction systems such as antirestriction proteins control RM systems. gene

9. Walkinshaw et al. Mol. Cell (2002) 9, 187. McMahon et al., (2009) Nucl. Acids Res. 37, 4887. Serfiotis-Mitsa et al., (2010) Nucl. Acids Res. 38, 1723. ArdA from transposon Tn916 Ocr from phage T7 ArdB from a B. pertussis plasmid Antirestriction systems such as antirestriction proteins control RM systems.

10. Bacterial DNA restriction and modification systems: structure, function and evolution. 1. Restriction and modification (RM) systems and their inhibition by antirestriction systems. 2. Type I RM enzymes: structure, function and evolution. 3. Some speculation on the antiquity of RM systems and their influence on evolution.

11. The components of Type I DNA RM enzymes. Kennaway et al. Genes and Development (2012) 26, 92-104. Type I RM enzyme comprises 5 subunits: R2M2S1 Molecular mass: 440,000 Highly conserved in sequence and structure except for target recognition domains in the S subunits.

12. CC group / enzyme Trd1spacerTrd2 CC1-1CCAY(N) 5 TTAA CC1-2CCAY(N) 6 TGT CC5-1ATC(N) 5 CCT CC5-2CCAY(N) 6 GTA CC133-771CAG(N) 5 RTGA CC398-1ACC(N) 5 RTGA 26 other CC12 others14 others HsdS Type I RM systems in S. aureus allow changes of specificity.

13. EM model for a Type I RM enzyme with DNA bound. HsdR (red), HsdM (blue and cyan), HsdS (yellow). Kennaway et al. Genes and Development (2012) 26, 92-104.

14. R NucleaseMotor M S promoter frameshift CCAYTTAA N-term catalytictail cr 5 M S promoter frameshift CCAYTGT N-term catalytictail cr 6 M S promoter frameshift ATCCCT N-term catalytictail cr 5 M S promoter frameshift CCAYGTA N-term catalytictail cr 6 CC1-1 CC5-2 promoter tail N-term CC1, CC5, CC8/ST239 HsdR HsdMHsdS Type I RM systems in S. aureus control horizontal gene transfer. Roberts et al. Nucl. Acids Res. doi: 10.1093/nar/gkt535

15. Bacterial DNA restriction and modification systems: structure, function and evolution. 1. Restriction and modification (RM) systems and their inhibition by antirestriction systems. 2. Type I RM enzymes: structure, function and evolution. 3. Some speculation on the antiquity of RM systems and their influence on evolution.

16. What was the first RM system? Williams. J. Inorg. Biochem. (2006), 100, 1908 What did the first RM system look like? When did it appear? What pressure caused it to diversify? (Antirestriction perhaps?)

17. Why was the first RM system not a Type II RM system? They are too specialised. Subtypes of Type II restriction enzymes. Type II restriction enzymes highly variable in sequence and structure but the methylases are conserved. Pingoud and Jeltsch. Nucl Acids Res (2001) 29, 3705 Cheng and Roberts. Nucl Acids Res (2001) 29, 3784

18. What was the first RM system? Perhaps it was a proto-Type I RM enzyme? The first RM system could be built from DNA repair enzymes, small-molecule methylases and transcription factors. These would be present before the LUCA. Most antirestriction targets Type I RM systems. This would force diversification in RM structure to avoid antirestriction DNA mimics and create new RM Types.

19. What would happen to Horizontal Gene Transfer with "perfect" restriction and modification systems or "perfect" antirestriction? SLIME Why did RM and anti-RM appear?