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

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Presentation on theme: "Early studies on the EcoB restriction enzyme using filamentous phage DNA Kensuke Horiuchi The Rockefeller University."— Presentation transcript:

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

2 Recognition site CleavedIntact Restriction Endonuclease BindsDoes not bind Me

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 e.o.p. on E.coli Ke.o.p. on E.coli B f1.K1.07 x f1.B1.0 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 PhageGenotypeNo. of SBe.o.p. on B Wild typeSB 1 + SB 2 + SB = 27 X One step mutantSB 1 + SB 2 0 SB = 13 X One step mutantSB 1 0 SB 2 + SB = 13 X Two step mutantSB 1 0 SB 2 0 SB = 01.0

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

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

9 EcoB does not cleave DNA at defined sites Horiuchi & Zinder (1972) PNAS 1)If EcoB cleaves f1 RF DNA at a single specific site, annealing after denaturation should yield only linear molecules. 2)If cleavage sites are not specific, reannealing should yield circular DNA and multimers. Mutant with a single SB site

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

11 Effect of fragmentation of lambda DNA on EcoB enzyme activity

12 1)EcoB recognizes DNA at SB sites. Recognition is independent of DNA length. 2)The probability that linear DNA is cleaved by bound enzyme depends on DNA length. 3)Circular DNA has an increased probability of cleavage. 4)Thus the enzyme likely needs to translocate along DNA before cleavage. 5)After DNA cleavage, the enzyme (or its components) remains on DNA and causes massive ATP hydrolysis. Steps in EcoB endonuclease action Horiuchi, Vovis & Zinder (1974) JBC

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

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

15 Ravetch, Horiuchi & Zinder (1978) PNAS

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

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

18 At the 50 th CSH Phage Meeting (1995)

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

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

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

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

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25 Genetic assay for DNA breaks

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


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