Presentation on theme: "Early studies on the EcoB restriction enzyme using filamentous phage DNA Kensuke Horiuchi The Rockefeller University."— Presentation transcript:
Early studies on the EcoB restriction enzyme using filamentous phage DNA Kensuke Horiuchi The Rockefeller University
Recognition site CleavedIntact Restriction Endonuclease BindsDoes not bind Me
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.
Norton raised the possibility that the cleavage site and the recognition site are distinct.
e.o.p. on E.coli Ke.o.p. on E.coli B f1.K1.07 x 10 -4 f1.B1.0 Phage f1 is restricted by EcoB but not by EcoK
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 10 -4 One step mutantSB 1 + SB 2 0 SB = 13 X 10 -2 One step mutantSB 1 0 SB 2 + SB = 13 X 10 -2 Two step mutantSB 1 0 SB 2 0 SB = 01.0
Lyons & Zinder (1972) Virology Genetic Map of f1
Horiuchi & Zinder (1972) PNAS Cleavage of f1 RFI by EcoB enzyme I supercoiled DNA II nicked circular DNA III linear DNA
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
ATP hydrolysis continues after DNA cleavage Horiuchi, Vovis & Zinder (1974) JBC
Effect of fragmentation of lambda DNA on EcoB enzyme activity
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
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
Physical map of f1 by type II restriction enzymes Hae III Hpa II Hha I Genes