Early studies on the EcoB restriction enzyme using filamentous phage DNA

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
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
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
6
F1 has two E. coli B sensitive sites
Phage Genotype No. of SB e.o.p. on B
Wild type SB1 SB2 SB 2 7 X 10-4
One step mutant SB1 SB20 SB 1 3 X 10-2
One step mutant SB10 SB2 SB 1 3 X 10-2
Two step mutant SB10 SB20 SB 0 1.0
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

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.

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

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.

Horiuchi, Vovis Zinder (1974) JBC
13
Methyl transfer activity of EcoB on
hemimethylated f1 RF
SB/SB -gt endonuclease SB/SBM -gt methyl
transferase SBM/SBM -gt 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 Models house in
1989
18
At the 50th CSH Phage Meeting (1995)
19
(No Transcript)
20
Four point cross genetic mapping of f1
Lyons Zinder (1972) Virology
21
ATP hydrolysis continues without new DNA-protein
interaction
Horiuchi, Vovis Zinder (1974) JBC
22
Inactive short linear DNA competes with long DNA
Horiuchi, Vovis Zinder (1974) JBC
23
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
24
(No Transcript)
25
Genetic assay for DNA breaks
26
Sites of f1 DNA scission by EcoRI star mutant
endonucleases
Heitman Model (1990) EMBO J.