early studies on the ecob restriction enzyme using filamentous phage dna

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