early studies on the ecob restriction enzyme using filamentous phage dna
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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. - PowerPoint PPT PresentationTRANSCRIPT
Early studies on the EcoB restriction enzyme using filamentous phage DNA
Kensuke HoriuchiThe Rockefeller University
Recognition site Recognition site
Cleaved Intact
Restriction Endonuclease
Binds Does 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 K e.o.p. on E.coli B
f1.K 1.0 7 x 10-4
f1.B 1.0 1.0
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
Phage Genotype No. of SB e.o.p. on B
Wild type SB1+ SB2
+ SB = 2 7 X 10-4
One step mutant SB1+ SB2
0 SB = 1 3 X 10-2
One step mutant SB10 SB2
+ SB = 1 3 X 10-2
Two step mutant SB10 SB2
0 SB = 0 1.0
Lyons & Zinder (1972) Virology
Genetic Map of f1
Horiuchi & Zinder (1972) PNAS
Cleavage of f1 RFI by EcoB enzyme
I supercoiled DNAII nicked circular DNAIII 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
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+ -> endonucleaseSB+/SBM -> methyl transferaseSBM/SBM -> no recognition
Physical map of f1 by type II restriction enzymes
Hae IIIHpa IIHha IGenes
Ravetch, Horiuchi & Zinder (1978) PNAS
Horiuchi & Zinder (1976) PNAS
Origin and direction of f1 DNA replication in vivo
A Zinder lab at a party at Peter Model’s house in 1989
At the 50th CSH Phage Meeting (1995)
Lyons & Zinder (1972) Virology
Four point cross: genetic mapping of f1
Horiuchi, Vovis & Zinder (1974) JBC
ATP hydrolysis continues without new DNA-protein interaction
Inactive short linear DNA competes with long DNA
Horiuchi, Vovis & Zinder (1974) JBC
Horiuchi & Zinder (1975) PNAS
A: RF cleavedB: RF cleaved + strandC: + strand cleaved
Site-specific cleavage of f1 single-stranded DNA by Hae III
Genetic assay for DNA breaks
Heitman & Model (1990) EMBO J.
Sites of f1 DNA scission by EcoRI star mutant endonucleases