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Early studies on the EcoB restriction enzyme using filamentous phage DNA

Early studies on the EcoB restriction enzyme using filamentous phage DNA. Kensuke Horiuchi The Rockefeller University. Restriction Endonuclease. Binds. Does not bind. Me. Recognition site. Recognition site. Cleaved. Intact. What we discovered about EcoB.

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Early studies on the EcoB restriction enzyme using filamentous phage DNA

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  1. Early studies on the EcoB restriction enzyme using filamentous phage DNA Kensuke Horiuchi The Rockefeller University

  2. Restriction Endonuclease Binds Does not bind Me Recognition site Recognition site Cleaved Intact

  3. What we discovered about EcoB • The cleavage site is different from the recognition site. • Cleavage does not occur at a defined site but occurs after the enzyme translocates along the DNA.

  4. Norton raised the possibility that the cleavage site and the recognition site are distinct.

  5. Phage f1 is restricted by EcoB but not by EcoK

  6. F1 has two E. coli B sensitive sites Arber & Kuehnlein (1969) Path. Microbiol. Boon & Zinder (1971) JMB

  7. Genetic Map of f1 Lyons & Zinder (1972) Virology

  8. Cleavage of f1 RFI by EcoB enzyme I supercoiled DNA II nicked circular DNA III linear DNA Horiuchi & Zinder (1972) PNAS

  9. EcoB does not cleave DNA at defined sites Mutant with a single SB site • If EcoB cleaves f1 RF DNA at a single specific site, annealing after denaturation should yield only linear molecules. • If cleavage sites are not specific, reannealing should yield circular DNA and multimers. Horiuchi & Zinder (1972) PNAS

  10. ATP hydrolysis continues after DNA cleavage Horiuchi, Vovis & Zinder (1974) JBC

  11. Effect of fragmentation of lambda DNA on EcoB enzyme activity Horiuchi, Vovis & Zinder (1974) JBC

  12. Steps in EcoB endonuclease action • EcoB recognizes DNA at SB sites. Recognition is independent of DNA length. • The probability that linear DNA is cleaved by bound enzyme depends on DNA length. • Circular DNA has an increased probability of cleavage. • Thus the enzyme likely needs to translocate along DNA before cleavage. • After DNA cleavage, the enzyme (or its components) remains on DNA and causes massive ATP hydrolysis. Horiuchi, Vovis & Zinder (1974) JBC

  13. Methyl transfer activity of EcoB on hemimethylated f1 RF SB+/SB+ -> endonuclease SB+/SBM -> methyl transferase SBM/SBM -> no recognition Vovis, Horiuchi & Zinder (1974) PNAS

  14. Physical map of f1 by type II restriction enzymes Hae III Hpa II Hha I Genes

  15. Ravetch, Horiuchi & Zinder (1978) PNAS

  16. Origin and direction of f1 DNA replication in vivo Horiuchi & Zinder (1976) PNAS

  17. A Zinder lab at a party at Peter Model’s house in 1989

  18. At the 50th CSH Phage Meeting (1995)

  19. Four point cross: genetic mapping of f1 Lyons & Zinder (1972) Virology

  20. ATP hydrolysis continues without new DNA-protein interaction Horiuchi, Vovis & Zinder (1974) JBC

  21. Inactive short linear DNA competes with long DNA Horiuchi, Vovis & Zinder (1974) JBC

  22. Site-specific cleavage of f1 single-stranded DNA by Hae III A: RF cleaved B: RF cleaved  + strand C: + strand cleaved Horiuchi & Zinder (1975) PNAS

  23. Genetic assay for DNA breaks

  24. Sites of f1 DNA scission by EcoRI star mutant endonucleases Heitman & Model (1990) EMBO J.

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