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Restriction Enzymes

Restriction Enzymes. Presented by: Elizabeth Gordon April 11,2006. Function. (From Tock and Dryden 2005). Historical Perspective. (From Lewin 2003). Four Types. (From Tock and Dryden 2005). Type I. Three subunits form R 2 M 2 S 1

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Restriction Enzymes

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  1. Restriction Enzymes Presented by: Elizabeth Gordon April 11,2006

  2. Function (From Tock and Dryden 2005)

  3. Historical Perspective (From Lewin 2003)

  4. Four Types (From Tock and Dryden 2005)

  5. Type I • Three subunits form R2M2S1 • Restriction endonuclease, methyltransferase, and ATPase activities • Requires ATP, SAM, and Mg2+ • Asymmetric, two-part sequence • Cleaves approximately 1KB away from recognition site • Can be used for DNA Translocation

  6. Type II • Two different subunits form R2 or M1 • Restriction endonuclease or methyltransferase • Requires SAM and Mg2+ • Mostly symmetric recognition site • Cleaves at or near site • No DNA translocation capacity

  7. Type III • Two different subunits form mod2res2 • Restriction endonuclease, methyltransferase, and ATPase activities • Requires ATP, SAM, and Mg2+ • Asymmetric • Cleaves 25-27 bp away from the recognition site • DNA translocation capability

  8. Type IV • Two subunits: McrB, McrC • Restriction endonuclease and GTPase • Requires GTP and Mg2+ • No methylation activity • Recognition sequence is two-part and methylated • Cleaves between methylated bases • Can assist in DNA translocation

  9. Homing Endonucleases • Used to create introns • Create double stranded breaks • Insert into intronless alleles • Cleave at a specific sequence • Recognize 12-40 bp sequences • Can form RNP complexes

  10. Red Queen Principle

  11. Anti-Restriction Strategies Pt 1 • Loss of restriction and methylation recognition sites • Strand-biased asymmetrical recognition sequences • Incorporation of unusual bases into phage genomes • Phage-encoded DNA-modifying enzymes

  12. Anti-Restriction Strategies Pt 2 • Stimulation of restriction-methylation system • Depletion of intracellular co-factors • Phage-encoded proteins • Plasmid-encoded proteins

  13. Future Research • Use of anti-restriction to treat bacterial pathogens • Studying the role of Homing Endonucleases in the creation of introns • Exploring any possible unknown categories of restriction enzymes

  14. Questions?

  15. Literature Cited • Belfort, M and Roberts, RJ. “Homing endonucleases: Keeping the house in order.” Nucleic Acids Research. 1997. 25, 17:3379-3388 • Tock, MR and Dryden, DTF. “The biology of restriction and anti-restriction.” Current Opinion in Microbiology. 2005. 8:466-472 • Lewin, B. Genes VII. Prentice Hall. 2003. • Van Valen L. “A New Evolutionary Law.” Evolutionary Theory 1973. 1, 1-30

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