Restriction Enzymes

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, 173379-3388
• Tock, MR and Dryden, DTF. The biology of
  restriction and anti-restriction. Current
  Opinion in Microbiology. 2005. 8466-472
• Lewin, B. Genes VII. Prentice Hall. 2003.
• Van Valen L. A New Evolutionary Law.
  Evolutionary Theory 1973. 1, 1-30