A

Switch

in

the

Mechanism

of

Communication

between

the

Two

DNA-Binding

Sites

in

the

SfiI

Restriction

Endonuclease

While

many

Type

II

restriction

enzymes

are

dimers

with

a

single

DNA-binding

cleft

between

the

subunits

SfiI

is

a

tetramer

of

identical

subunits

Two

of

its

subunits

-LRB-

a

dimeric

unit

-RRB-

create

one

DNA-binding

cleft

and

the

other

two

create

a

second

cleft

on

the

opposite

side

of

the

protein

The

two

clefts

bind

specific

DNA

cooperatively

to

give

a

complex

of

SfiI

with

two

recognition

sites

This

complex

is

responsible

for

essentially

all

of

the

DNA-cleavage

reactions

by

SfiI

virtually

is

due

to

the

complex

with

one

site

The

communication

between

the

DNA-binding

clefts

was

examined

by

disrupting

one

of

the

very

few

polar

interactions

in

the

otherwise

hydrophobic

interface

between

the

dimeric

units

a

tyrosine

hydroxyl

was

removed

by

mutation

to

phenylalanine

The

mutant

protein

remained

tetrameric

in

solution

and

could

bind

two

DNA

sites

But

instead

of

being

activated

by

binding

two

sites

like

wild-type

SfiI

it

showed

maximal

activity

when

bound

to

a

single

site

and

had

a

lower

activity

when

bound

to

two

sites

This

interaction

across

the

dimer

interface

thus

enforces

in

wild-type

SfiI

a

cooperative

transition

between

inactive

and

active

states

in

both

dimers

but

without

this

interaction

as

in

the

mutant

protein

a

single

dimer

can

undergo

the

transition

to

give

a

stable

intermediate

with

one

inactive

dimer

and

one

active

dimer

Introduction

Type

II

restriction

endonucleases

recognise

specific

sequences

in

DNA

typically

palindromic

sites

to

bp

long

and

cut

the

DNA

at

specified

positions

within

or

close

to

the

site

Their

reactions

usually

-LRB-

but

not

-RRB-

require

as

a

cofactor

Some

Type

II

endonucleases

are

dimers

of

identical

subunits

that

interact

symmetrically

with

their

palindromic

sites

These

have

a

single

DNA-binding

cleft

at

the

subunit

interface

and

they

act

at

individual

copies

of

their

target

sites

The

best-studied

restriction

enzymes

such

as

EcoRV

and

BamHI

and

those

most

widely

used

in

vitro

as

tools

for

molecular

biology

all

function

in

this

manner

Consequently

these

are

often

considered

as

standard

restriction

enzymes

However

many

Type

II

endonucleases

differ

from

the

standard

in

that

they

are

fully

active

only

after

interacting

with

two

copies

of

their

recognition

site

The

Type

II

enzymes

that

need

two

sites

fall

into

two

subtypes

IIE

or

IIF

The

Type

IIE

restriction

enzymes

bind

two

-LRB-

or

-RRB-

copies

of

their

recognition

sequence

yet

cleave

only

one

They

contain

two

DNA-binding

clefts

catalytic

and

allosteric

but

the

catalytic

cleft

is

inactive

unless

cognate

DNA

is

also

bound

at

the

allosteric

site

In

contrast

the

Type

IIF

enzymes

form

complexes

with

two

DNA

sites

at

equivalent

loci

in

the

protein

and

then

cut

both

sites

in

a

concerted

reaction

The

first

restriction

enzyme

found

to

act

concertedly

at

two

DNA

sites

was

the

SfiI

endonuclease

SfiI

recognises

the

sequence

GGCCNNNN

NGGCC

-LRB-

where

N

is

any

base

and

is

the

point

of

cleavage

-RRB-

but

it

cleaves

substrates

with

two

copies

of

this

sequence

more

rapidly

than

DNA

with

a

single

copy

Moreover

in

steady-state

reactions

at

low

enzyme

concentrations

SfiI

converts

DNA

with

two

sites

directly

to

the

final

product

cut

at

both

sites

without

liberating

intermediates

cut

at

one

site

the

intermediates

remain

bound

to

the

enzyme

until

it

has

cut

both

strands

at

both

sites

On

DNA

with

two

sites

SfiI

loops

out

the

DNA

between

the

sites

but

it

can

also

bind

simultaneously

to

two

separate

DNA

molecules

that

each

have

one

copy

of

the

recognition

sequence

The

rates

of

their

reactions

on

one-site

substrates

increase

sigmoidally

with

DNA

concentration

indicative

of

positive

cooperativity

The

interaction

with

two

sites

is

obligatory

as

virtually

no

DNA

is

cleaved

by

any

complex

of

SfiI

with

a

single

recognition

site

Many

genetic

events

such

as

DNA

replication

and

recombination

and

the

regulation

of

gene

expression

often

depend

on

proteins

interacting

with

two

sites

at

separate

locations

in

the

DNA

and

the

SfiI

restriction

enzyme

has

become

one

of

the

principal

test

systems

for

analysing

the

mechanisms

of

long-range

communications

between

distant

DNA

sites

Many

restriction

enzymes

are

now

known

to

belong

to

the

Type

IIF

family

examples

include

NgoMIV

BspMI

SgrAI

and

BbeI

and

almost

all

of

the

Type

II

enzymes

that

cut

DNA

bilaterally

on

either

side

of

their

recognition

sites

such

as

BcgI

and

AloI

All

of

these

enzymes

need

to

interact

with

two

recognition

sites

for

full

activity

Proteins

that

interact

with

two

DNA

sites

generally

prefer

sites

in

cis

on

the

same

molecule

of

DNA

to

sites

in

trans

on

separate

DNA

molecules

simply

because

two

sites

in

cis

will

almost

always

be

in

closer

proximity

than

sites

in

trans

Consequently

all

of

these

enzymes

are

capable

of

cleaving

DNA

with

two

sites

more

rapidly

than

DNA

with

one

site

Most

Type

IIF

enzymes

including

SfiI

are

known

from

analytical

ultracentrifugation

-LRB-

AUC

-RRB-

studies

to

exist

in

solution

as

tetramers

of

identical

subunits

-LSB-

SgrAI

is

an

exception

but

while

it

is

a

dimer

in

solution

two

dimers

bound

to

separate

sites

associate

to

form

a

tetramer

before

cutting

DNA

-RSB-

In

the

crystal

structures

of

the

tetrameric

enzymes

two

of

the

subunits

-LRB-

a

primary

dimer

-RRB-

constitute

one

DNA-binding

cleft

and

the

other

two

subunits

constitute

a

second

identical

cleft

The

two

primary

dimers

are

arranged

back-to-back

so

that

their

DNA-binding

clefts

are

on

the

opposite

sides

of

the

protein

-LRB-

Figure

-LRB-

a

-RRB-

and

-LRB-

b

-RRB-

-RRB-

The

primary

dimers

in

SfiI

are

comparable

to

the

dimeric

restriction

enzyme

BglI

an

enzyme

whose

recognition

site

GCCNNNN

NGGC

is

a

truncated

SfiI

site

The

structures

of

the

individual

subunits

of

SfiI

and

the

arrangement

of

the

two

subunits

in

its

primary

dimer

are

similar

to

those

in

BglI

likewise

its

motifs

for

DNA

sequence

recognition

and

catalysis

The

subunit

interface

within

the

dimer

has

however

a

much

smaller

area

in

SfiI

than

in

BglI

In

addition

SfiI

and

BglI

have

very

different

surfaces

opposite

the

DNA-binding

cleft

polar

and

solvent-exposed

in

the

case

of

dimeric

BglI

but

almost

completely

hydrophobic

and

buried

in

tetrameric

SfiI

In

previous

studies

the

mode

of

communication

between

the

two

DNA-binding

clefts

in

another

tetrameric

Type

IIF

enzyme

was

examined

by

mutating

selected

amino

acids

at

the

dimer

dimer

interface

A

mutation

that

converted

the

tetramer

into

a

dimer

yielded

an

enzyme

with

much

the

same

properties

as

a

standard

dimeric

restriction

enzyme

while

wild-type

-LRB-

wt

-RRB-

cleaves

DNA

with

two

target

sites

at

a

rapid

rate

and

DNA

with

one

site

at

a

slow

rate

the

dimeric

mutant

cleaved

both

substrates

at

equally

rapid

rates

Another

mutant

at

the

dimer

interface

of

cleaved

both

one-site

and

two-site

substrates

at

diminished

rates

while

a

third

cleaved

both

substrates

at

elevated

rates

but

in

both

cases

the

two-site

DNA

was

still

cleaved

more

rapidly

than

the

one-site

DNA

In

this

study

we

report

on

the

effect

of

a

single

amino-acid

substitution

at

the

dimer

dimer

interface

of

the

SfiI

endonuclease

a

conservative

change

from

a

tyrosine

to

a

phenylalanine

that

removes

just

one

hydroxyl

group

per

subunit

In

contrast

to

the

previous

mutants

of

this

mutation

yields

an

enzyme

that

cleaves

DNA

with

one

cognate

site

more

rapidly

than

DNA

with

two

sites

It

thus

switches

SfiI

from

an

enzyme

that

is

activated

by

binding

two

DNA

sites

to

one

that

shows

its

maximal

activity

on

binding

a

single

site

Results

Subunit

communications

in

SfiI

In

the

crystal

structure

of

SfiI

bound

to

two

copies

of

its

recognition

sequence

two

subunits

-LRB-

a

primary

dimer

-RRB-

bind

one

duplex

with

each

monomer

contacting

one

of

the

two

GGCC

elements

of

the

sequence

The

other

two

subunits

bind

the

second

duplex

on

the

opposite

side

of

the

tetramer

in

a

back-to-back

arrangement

-LRB-

Figure

-LRB-

a

-RRB-

and

-LRB-

b

-RRB-

-RRB-

The

interface

between

the

two

monomers

within

each

dimer

has

a

surface

area

-LRB-

Å2

-RRB-

that

is

not

only

smaller

than

that

for

the

related

dimeric

enzyme

BglI

-LRB-

Å2

-RRB-

but

which

is

also

smaller

than

those

for

other

tetrameric

restriction

enzymes

-LRB-

viz.

Å2

for

-RRB-

Many

of

the

amino

acids

at

the

interface

within

each

dimer

are

polar

in

nature

and

interact

with

the

opposite

monomer

In

contrast

the

subunit

interactions

at

the

interface

between

the

primary

dimers

have

a

larger

contact

area

in

SfiI

than

in

the

other

tetramers

-LRB-

Å2

compared

to

Å2

for

-RRB-

In

SfiI

the

inter-dimer

interface

is

composed

almost

entirely

of

nonpolar

residues

the

amino

acids

in

one

subunit

that

lie

within

Å

of

the

opposite

subunit

across

the

dimer

interface

-LRB-

Figure

-LRB-

c

-RRB-

-RRB-

coincide

with

a

patch

on

this

surface

that

is

devoid

of

either

positively

or

negatively

charged

side

-LRB-

Figure

-LRB-

d

-RRB-

-RRB-

The

subunit

interactions

across

the

inter-dimer

interface

are

thus

mainly

van

der

Waals

contacts

between

hydrophobic

side

chains

although

they

also

include

some

contacts

between

peptidyl

main-chain

carbonyl

and

amino

groups

However

there

appear

to

be

only

two

side-chain-to-side-chain

hydrogen

bonds

across

the

inter-dimer

interface

between

in

one

subunit

and

in

the

opposite

partner

likewise

between

and

-LRB-

Figure

-LRB-

c

-RRB-

-RRB-

is

located

at

the

outside

edge

of

the

inter-dimer

interface

-LRB-

Figure

-LRB-

c

-RRB-

-RRB-

while

is

positioned

near

the

centre

of

the

contact

area

-LRB-

Figure

-LRB-

c

-RRB-

and

-LRB-

d

-RRB-

-RRB-

Moreover

the

aromatic

side

chain

of

protrudes

from

the

surface

of

each

subunit

into

a

pocket

in

the

opposite

subunit

-LRB-

Figure

-LRB-

b

-RRB-

-RRB-

Given

the

paucity

of

directional

interactions

across

this

interface

it

seemed

plausible

that

might

play

a

pivotal

role

in

the

communication

between

the

two

DNA-binding

clefts

To

test

this

possibility

the

hydroxyl

moiety

from

the

side

chain

was

removed

by

using

site-directed

mutagenesis

to

replace

the

tyrosine

with

phenylalanine

to

yield

the

mutant

Enzyme

stability

and

quaternary

structure

DNA-cleavage

reactions

of

the

mutant

were

initially

carried

out

at

°C

-LRB-

the

standard

temperature

for

SfiI

assays

-RRB-

in

the

same

way

as

for

wt

SfiI

the

enzyme

was

diluted

into

dilution

buffer

-LRB-

Materials

and

Methods

-RRB-

incubated

at

°C

and

then

added

to

a

solution

of

DNA

and

However

lost

activity

rapidly

between

dilution

and

subsequent

addition

to

the

DNA

after

and

min

in

dilution

buffer

at

°C

its

activity

had

fallen

by

factors

of

and

respectively

while

the

wt

enzyme

retained

full

activity

-LRB-

data

not

shown

-RRB-

However

when

the

enzyme

was

mixed

with

the

DNA

substrate

before

initiating

the

reaction

with

it

retained

full

activity

For

wt

SfiI

no

differences

were

observed

between

reactions

initiated

by

adding

diluted

enzyme

to

the

mix

of

DNA

and

and

reactions

initiated

by

adding

to

premixed

enzyme

and

DNA

All

subsequent

analyses

of

were

therefore

carried

out

in

the

presence

of

cognate

DNA

and

all

DNA-cleavage

reactions

were

initiated

by

adding

to

mixtures

of

enzyme

and

DNA

Since

the

protein

is

unstable

in

the

absence

of

DNA

its

oligomeric

state

was

determined

with

DNA

present

The

DNA

was

a

duplex

with

the

recognition

sequence

for

SfiI

-LRB-

Table

-RRB-

This

duplex

carries

a

chromophoric

label

hexachlorofluorescein

-LRB-

HEX

-RRB-

attached

to

the

end

of

the

top

strand

has

an

absorbance

peak

at

nm

a

wavelength

at

which

neither

protein

nor

nucleotides

have

any

intrinsic

absorbance

The

sedimentation

of

to

equilibrium

was

measured

by

recording

in

the

AUC

the

absorbance

of

the

samples

at

nm

as

a

function

of

centrifugal

radius

The

samples

comprised

alone

or

with

either

or

wt

SfiI

-LRB-

the

DNA

was

present

at

half

the

concentration

of

DNA-binding

sites

in

the

enzyme

-RRB-

In

all

three

cases

the

increase

in

absorbance

with

centrifugal

radius

was

fitted

to

the

equation

for

a

single

homogenous

to

yield

apparent

Mr

-LRB-

relative

molecular

mass

-RRB-

values

By

itself

gave

an

Mr

of

about

close

to

that

expected

for

a

HEX-labelled

DNA

of

bp

-LRB-

data

not

shown

-RRB-

The

best

fits

to

the

data

in

the

presence

of

wt

SfiI

or

gave

Mr

values

of

and

respectively

-LRB-

Figure

-RRB-

which

are

both

close

to

that

predicted

for

the

SfiI

tetramer

bound

to

two

duplexes

-LRB-

-RRB-

The

removal

of

the

hydroxyl

group

from

the

side

chain

of

by

replacing

it

with

Phe

diminished

the

stability

of

the

protein

upon

dilution

to

low

protein

concentrations

the

mutant

lost

activity

possibly

due

to

subunit

dissociation

at

low

concentrations

Nevertheless

as

with

wt

SfiI

the

Mr

of

bound

to

a

DNA

matched

that

expected

for

the

tetramer

bound

to

two

duplexes

DNA-binding

studies

Gel

retardation

was

used

to

compare

the

DNA

protein

complexes

formed

by

and

by

wt

SfiI

-LRB-

Figure

-RRB-

As

in

previous

studies

two

duplexes

of

different

lengths

were

employed

the

DNA

used

in

the

AUC

and

an

elongated

version

-LRB-

Table

-RRB-

The

complexes

of

enzyme

bound

to

either

duplex

or

to

both

were

separated

from

each

other

and

from

the

free

duplexes

by

electrophoresis

through

polyacrylamide

The

DNA

was

detected

by

HEX

fluorescence

The

two

duplexes

were

added

at

a

constant

total

concentration

to

fixed

amounts

of

either

or

wt

SfiI

to

give

mixtures

that

contained

twice

the

molarity

of

duplex

over

enzyme

tetramer

The

binding

contained

this

ion

can

promote

specific

binding

by

dependent

enzymes

without

supporting

DNA

cleavage

The

addition

of

alone

to

either

protein

gave

a

single

retarded

complex

as

did

the

addition

of

alone

-LRB-

Figure

left-hand

and

right-hand

lanes

respectively

-RRB-

The

complex

with

had

a

faster

electrophoretic

mobility

than

that

with

When

both

duplexes

were

added

three

complexes

were

observed

one

with

the

same

mobility

as

that

with

one

equal

to

the

complex

with

and

a

third

with

an

intermediate

mobility

-LRB-

Figure

central

lanes

-RRB-

The

yields

of

the

three

complexes

varied

with

the

ratio

of

the

two

duplexes

in

a

binomial

manner

with

the

distribution

occurring

when

the

ratio

of

the

concentrations

of

to

was

close

to

The

same

pattern

was

observed

with

The

intermediate

complex

with

a

mobility

in

between

that

with

alone

and

that

with

alone

must

contain

one

molecule

of

and

one

molecule

of

The

complexes

with

the

highest

and

the

lowest

mobilities

therefore

correspond

respectively

to

the

SfiI

tetramer

bound

to

two

molecules

of

and

to

two

molecules

of

Both

and

wt

SfiI

can

thus

form

complexes

that

contain

two

DNA

duplexes

bound

to

a

tetrameric

protein

However

both

proteins

were

saturated

at

the

DNA

concentrations

used

here

so

it

remains

to

be

determined

whether

there

are

any

differences

in

affinity

and/or

cooperativity

between

wt

and

mutant

proteins

Cleavage

of

two-site

plasmid

The

optimal

substrates

for

SfiI

are

supercoiled

-LRB-

SC

-RRB-

plasmids

that

have

two

copies

of

its

recognition

sequence

A

single

tetramer

of

SfiI

binds

to

two

sites

in

cis

and

traps

the

intervening

DNA

in

a

loop

It

then

cleaves

all

four

of

its

target

phosphodiester

bonds

before

dissociating

from

the

DNA

the

initial

product

liberated

from

the

enzyme

is

the

final

product

cut

in

both

strands

at

both

sites

to

the

virtual

exclusion

of

intermediates

cut

at

one

two

or

three

bonds

To

find

out

if

the

mutant

acts

like

wt

SfiI

in

this

respect

the

reactions

of

both

enzymes

were

examined

on

a

plasmid

with

two

SfiI

sites

The

reactions

were

initially

conducted

under

steady-state

conditions

with

-LSB-

-RSB-

-LSB-

S

-RSB-

-LRB-

Figure

-LRB-

a

-RRB-

and

-LRB-

b

-RRB-

-RRB-

to

be

able

to

observe

directly

the

nature

of

the

DNA

liberated

from

the

enzyme

rather

than

the

enzyme-bound

intermediates

generated

during

the

reaction

Under

these

conditions

wt

SfiI

converted

almost

all

of

the

SC

plasmid

directly

to

the

two

linear

products

-LRB-

and

-RRB-

cut

in

both

strands

at

both

sites

of

the

open

circle

-LRB-

OC

-RRB-

form

of

the

DNA

cut

in

one

strand

and

only

a

small

amount

of

the

full-length

linear

-LRB-

LIN

-RRB-

form

of

the

DNA

cut

in

both

strands

at

one

site

were

released

from

the

enzyme

during

the

reaction

-LRB-

Figure

-LRB-

a

-RRB-

-RRB-

To

allow

comparisons

between

substrates

with

one

SfiI

site

and

substrates

with

two

SfiI

sites

all

steady-state

rates

are

recorded

here

in

terms

of

moles

of

SfiI

sites

cleaved

per

mole

of

enzyme

tetramer

per

minute

-LRB-

mol/mol/min

-RRB-

The

velocity

of

wt

SfiI

on

this

two-site

plasmid

was

mol/mol/min

-LRB-

Figure

-LRB-

a

-RRB-

-RRB-

the

Vmax

for

this

reaction

The

mutant

also

cleaved

the

two-site

plasmid

in

a

highly

concerted

manner

it

liberated

very

little

of

either

the

nicked

OC

form

or

the

LIN

product

cut

at

one

site

Instead

it

converted

this

DNA

directly

to

the

final

products

cut

at

both

sites

and

-LRB-

Figure

-LRB-

b

-RRB-

-RRB-

However

the

rate

at

which

cleaved

-LRB-

mol/mol/min

-RRB-

was

times

slower

than

wt

SfiI

It

is

unlikely

that

the

difference

is

due

to

a

difference

in

Km

as

the

gel-shift

experiments

-LRB-

Figure

-RRB-

had

shown

that

the

DNA

concentrations

used

here

were

sufficient

to

saturate

either

enzyme

However

the

rate-limiting

step

for

the

complete

reaction

pathway

for

wt

SfiI

-LRB-

the

process

that

determines

its

steady-state

velocity

-RRB-

is

the

final

dissociation

of

the

products

cut

at

both

sites

all

of

the

preceding

stages

including

the

four

DNA-cleavage

steps

are

relatively

rapid

The

reduction

in

reaction

velocity

could

thus

be

due

to

either

product

release

from

being

slower

than

from

wt

or

the

rates

of

the

DNA-cleavage

steps

in

its

reaction

pathway

being

reduced

to

values

below

that

for

product

release

by

wt

SfiI

These

possibilities

can

be

distinguished

by

determining

whether

generates

a

pre-steady

state

burst

of

product

formation

If

the

DNA

cleavage

steps

are

faster

than

product

release

the

enzyme

will

create

a

burst

of

enzyme-bound

product

to

a

concentration

equal

to

that

of

the

enzyme

prior

to

a

slow

steady-state

phase

the

rate

of

which

is

limited

by

product

release

On

the

other

hand

if

the

DNA-cleavage

steps

are

rate-limiting

for

there

will

be

no

burst

and

the

reaction

will

proceed

instead

at

the

linear

steady-state

rate

from

time

zero

The

reactions

of

both

wt

and

mutant

enzymes

were

studied

under

conditions

where

the

presence

of

a

burst

phase

is

readily

detected

with

an

enzyme

concentration

-LRB-

nM

-RRB-

approaching

that

of

the

DNA

-LRB-

nM

-RRB-

so

that

the

enzyme-bound

product

can

constitute

a

significant

fraction

of

the

total

product

and

at

a

reaction

temperature

-LRB-

°C

-RRB-

where

SfiI

has

a

very

slow

turnover

thus

allowing

the

reactions

at

elevated

enzyme

concentrations

to

still

be

monitored

Under

these

conditions

both

wt

and

enzymes

cleaved

a

fraction

of

the

two-site

substrate

rapidly

before

entering

a

slower

phase

during

which

the

concentration

of

the

substrate

declined

linearly

with

time

the

decline

was

more

rapid

with

wt

SfiI

than

with

-LRB-

Figure

-LRB-

c

-RRB-

-RRB-

Hence

on

a

DNA

with

two

sites

both

enzymes

are

rate-limited

by

product

release

but

releases

the

doubly

cut

product

more

slowly

than

wt

SfiI

-LSB-

For

both

enzymes

the

amount

of

substrate

consumed

in

the

initial

burst

phase

was

about

of

the

enzyme

concentration

rather

than

this

was

as

expected

as

the

addition

of

SfiI

to

a

two-site

substrate

leads

not

only

to

DNA

with

one

SfiI

tetramer

bridging

the

two

sites

in

cis

but

also

to

DNA

carrying

a

tetramer

at

each

site

which

resists

cleavage

-RSB-

Cleavage

of

one-site

plasmid

The

turnover

rate

of

wt

SfiI

on

a

plasmid

with

one

site

is

typically

about

times

slower

than

on

a

plasmid

with

two

sites

To

see

if

the

mutant

behaved

like

wt

both

enzymes

were

tested

against

a

plasmid

that

carries

one

of

the

two

SfiI

sites

from

During

these

reactions

the

SC

substrate

was

cleaved

directly

to

the

final

product

LIN

DNA

with

a

double-strand

break

at

the

SfiI

site

without

liberating

the

nicked

OC

form

-LRB-

data

not

shown

-RRB-

only

the

decline

in

the

substrate

concentration

is

shown

here

-LRB-

Figure

-RRB-

The

first

tests

employed

the

same

enzyme

concentration

-LRB-

nM

Figure

-LRB-

b

-RRB-

-RRB-

as

that

used

for

the

steady-state

reactions

on

the

two-site

plasmid

-LRB-

Figure

-LRB-

a

-RRB-

and

-LRB-

b

-RRB-

-RRB-

For

wt

SfiI

most

of

the

reaction

proceeded

with

a

linear

decline

in

the

concentration

of

the

substrate

with

time

and

as

expected

its

reaction

velocity

was

lower

than

that

on

the

two-site

plasmid

-LRB-

compared

to

mol/mol/min

-RRB-

However

the

same

concentration

of

yielded

markedly

biphasic

kinetics

a

rapid

phase

that

could

be

fitted

to

a

single

exponential

to

give

a

rate

constant

of

min

followed

by

a

slower

linear

phase

with

a

velocity

of

mol/mol/min

-LRB-

Figure

-LRB-

b

-RRB-

-RRB-

The

amount

of

substrate

consumed

during

the

fast

phase

-LRB-

about

nM

-RRB-

was

larger

than

the

amount

of

enzyme

in

this

reaction

-LRB-

nM

-RRB-

so

the

fast

phase

can

not

be

due

to

a

pre-steady-state

burst

of

product

formation

stoichiometric

with

the

enzyme

Instead

the

fast

phase

must

reflect

multiple

turnovers

of

an

active

form

of

the

enzyme

that

decays

exponentially

to

a

less

active

form

the

latter

being

responsible

for

the

slow

linear

phase

of

the

reaction

The

initial

rate

for

the

utilisation

of

the

one-site

plasmid

by

in

the

fast

phase

was

about

sevenfold

faster

than

its

reaction

on

the

two-site

plasmid

Moreover

even

during

the

slow

linear

phase

gave

virtually

the

same

rates

on

the

one-site

and

two-site

plasmids

-LRB-

and

mol/mol/min

respectively

-RRB-

To

see

whether

the

biphasicity

varied

with

enzyme

concentrations

both

and

wt

SfiI

were

tested

against

the

one-site

plasmid

at

lower

-LRB-

nM

Figure

-LRB-

a

-RRB-

-RRB-

and

at

higher

-LRB-

nM

Figure

-LRB-

c

-RRB-

-RRB-

concentrations

At

each

level

of

wt

SfiI

the

majority

of

the

SC

substrate

was

utilised

at

a

single

zero-order

rate

the

rates

increased

in

direct

proportion

to

the

enzyme

concentration

Low

concentrations

of

also

gave

for

most

of

the

reaction

a

linear

decline

in

the

concentration

of

the

one-site

substrate

with

time

at

a

velocity

-LRB-

mol/mol/min

Figure

-LRB-

a

-RRB-

-RRB-

that

was

similar

to

the

slow

phase

of

the

reaction

at

nM

-LRB-

mol/mol/min

Figure

-LRB-

b

-RRB-

-RRB-

In

contrast

at

high

levels

of

-LRB-

Figure

-LRB-

c

-RRB-

-RRB-

virtually

all

of

the

one-site

substrate

was

consumed

rapidly

at

a

similar

rate

to

the

fast

phase

from

the

reaction

with

nM

protein

The

active

form

of

that

gives

rise

to

the

rapid

phase

of

cutting

the

DNA

with

one

site

is

possibly

the

tetrameric

enzyme

bound

to

a

single

copy

of

its

recognition

sequence

This

then

may

decay

to

a

less

active

form

upon

binding

a

second

copy

to

give

the

synaptic

complex

the

tetramer

bound

to

two

recognition

sites

The

synaptic

complex

will

form

more

readily

on

a

two-site

DNA

than

on

a

one-site

DNA

so

both

wt

and

mutant

enzymes

are

likely

to

cleave

DNA

with

two

sites

by

forming

a

synaptic

complex

bridging

two

sites

in

cis

However

despite

the

difficulty

of

synapsing

sites

in

trans

wt

SfiI

cleaves

DNA

with

one

site

mainly

by

forming

synaptic

complexes

between

sites

in

separate

DNA

molecules

and

not

by

acting

at

individual

sites

The

complex

of

wt

SfiI

bound

to

one

site

is

therefore

either

inactive

or

never

present

at

a

significant

level

The

mutant

may

differ

from

wt

in

one

or

both

of

these

aspects

Reactions

in

trans

If

a

restriction

enzyme

cleaves

a

plasmid

with

a

single

target

by

acting

at

that

target

alone

then

the

addition

of

an

oligoduplex

carrying

the

recognition

sequence

will

reduce

the

rate

at

which

that

enzyme

cleaves

the

plasmid

as

the

duplex

will

act

as

a

competitive

inhibitor

On

the

other

hand

a

restriction

enzyme

that

cleaves

plasmids

with

a

single

site

by

spanning

two

sites

in

trans

will

be

activated

by

adding

an

appropriate

concentration

of

the

specific

duplex

as

the

synaptic

complex

can

then

be

formed

more

readily

with

one

molecule

of

plasmid

and

one

molecule

of

duplex

rather

than

with

two

molecules

of

plasmid

-LSB-

Higher

concentrations

of

duplex

will

however

lead

to

synaptic

complexes

with

two

molecules

of

the

duplex

and

thus

inhibit

plasmid

cleavage

-RSB-

Hence

if

the

fast

phase

of

the

reaction

of

is

due

to

its

reaction

at

an

individual

site

it

should

be

inhibited

by

adding

even

low

concentrations

of

cognate

duplex

But

if

it

is

due

to

a

reaction

in

trans

spanning

separate

molecules

of

DNA

it

should

be

activated

by

low

levels

of

the

duplex

Previous

experiments

had

shown

that

nM

duplex

activated

wt

SfiI

against

nM

one-site

plasmid

This

amount

of

duplex

was

therefore

added

to

the

reaction

of

on

a

one-site

plasmid

and

for

comparison

the

wt

reaction

-LRB-

Figure

-RRB-

-LRB-

Table

-RRB-

was

again

used

as

the

duplex

Relatively

high

enzyme

concentrations

were

employed

so

that

cleaved

almost

all

of

the

DNA

in

the

fast

phase

-LRB-

Figure

-LRB-

c

-RRB-

-RRB-

The

plasmid

with

one

site

was

cleaved

by

wt

SfiI

more

rapidly

in

the

presence

of

than

in

its

absence

-LRB-

Figure

-LRB-

a

-RRB-

-RRB-

In

marked

contrast

the

fast

phase

of

the

reaction

of

on

the

one-site

plasmid

was

much

slower

in

the

presence

of

than

in

its

absence

-LRB-

Figure

-LRB-

b

-RRB-

-RRB-

In

further

experiments

at

this

high

concentration

of

activated

cleavage

of

the

one-site

plasmid

was

not

observed

at

any

level

of

duplex

tested

-LRB-

from

nM

to

μM

-RRB-

However

at

nM

an

enzyme

concentration

that

results

in

most

of

the

one-site

plasmid

being

cleaved

in

the

slow

phase

-LRB-

Figure

-LRB-

a

-RRB-

-RRB-

the

addition

of

enhanced

the

reaction

rate

-LRB-

data

not

shown

-RRB-

It

thus

seems

likely

that

the

fast

phase

of

the

reaction

of

on

the

plasmid

with

one

SfiI

site

is

indeed

due

to

the

tetrameric

protein

bound

to

an

individual

site

while

the

slow

phase

of

the

reaction

and

the

monophasic

reaction

of

wt

SfiI

on

the

one-site

DNA

are

both

due

to

a

tetramer

bound

to

two

sites

in

trans

If

so

the

mutant

bound

to

one

SfiI

site

gives

a

much

higher

reaction

velocity

than

wt

SfiI

at

a

solitary

site

However

if

a

reaction

in

free

solution

is

carried

out

by

adding

a

one-site

DNA

to

a

protein

that

can

bind

two

sites

then

as

there

exists

no

physical

means

to

prevent

the

protein

from

binding

two

separate

molecules

of

DNA

one

can

never

completely

exclude

the

possibility

that

the

observed

reaction

involves

the

protein

bridging

sites

in

trans

Reactions

at

individual

sites

To

determine

unequivocally

whether

a

protein

capable

of

binding

two

DNA

sites

is

active

after

binding

just

one

the

protein

must

be

barred

from

contacting

two

DNA

molecules

at

the

same

time

This

can

be

achieved

by

immobilising

the

DNA

on

a

solid

surface

at

a

low

density

so

as

to

hold

the

DNA

molecules

separate

from

each

other

This

strategy

was

applied

to

SfiI

by

using

a

duplex

with

a

single

cognate

site

-LRB-

Table

-RRB-

The

site

in

this

duplex

is

the

same

as

that

in

the

one-site

plasmid

with

respect

to

spacer

and

flanking

sequences

The

duplex

carried

a

biotin

tag

on

the

end

of

one

strand

and

this

was

used

to

attach

it

to

a

streptavidin-coated

bead

The

ratio

of

streptavidin

on

the

bead

to

biotinylated

DNA

was

set

at

conditions

that

result

in

the

mean

distance

between

adjacent

DNA

molecules

on

the

surface

of

the

bead

being

considerably

longer

than

that

between

the

two

DNA-binding

clefts

in

the

SfiI

tetramer

This

leaves

the

individual

DNA

chains

too

far

apart

to

allow

SfiI

to

form

a

synaptic

complex

with

two

duplexes

The

duplex

also

contained

a

label

at

the

end

of

the

other

strand

so

that

cleavage

of

this

strand

could

be

monitored

These

experiments

employed

enzyme

concentrations

in

excess

of

immobilised

DNA

and

thus

are

effectively

single-turnover

reactions

The

observed

rates

of

DNA

cleavage

thus

reflect

the

phosphodiester

hydrolysis

step

in

the

reaction

pathway

Even

though

wt

SfiI

generally

acts

on

DNA

substrates

with

a

single

site

by

bridging

two

DNA

molecules

in

trans

-LRB-

Figures

-LRB-

a

-RRB-

and

-LRB-

a

-RRB-

-RRB-

it

was

still

able

to

cleave

a

one-site

DNA

even

when

the

individual

molecules

of

that

DNA

were

isolated

from

each

other

by

immobilisation

on

the

bead

-LRB-

Figure

-RRB-

The

extent

of

cleavage

of

the

immobilised

substrate

by

wt

SfiI

was

fitted

to

a

single

exponential

to

give

an

apparent

rate

constant

of

min

This

rate

constant

for

phosphodiester

hydrolysis

by

wt

SfiI

at

a

single

site

is

very

much

slower

than

measured

previously

for

the

same

process

in

its

synaptic

complex

with

two

sites

-LRB-

s

-RRB-

Even

though

the

immobilised

duplex

used

here

differs

from

the

plasmids

used

previously

for

reactions

in

bulk

solution

this

difference

is

consistent

with

other

studies

that

have

shown

that

the

activity

of

wt

SfiI

bound

to

two

sites

is

times

higher

than

that

at

a

single

site

The

single-turnover

reaction

of

on

the

immobilised

DNA

with

one

SfiI

site

also

gave

an

exponential

decline

in

substrate

concentration

with

an

apparent

rate

constant

of

min

-LRB-

Figure

-RRB-

much

faster

than

wt

SfiI

Hence

the

mutant

bound

to

a

single

site

cleaves

that

site

more

rapidly

than

wt

SfiI

at

an

individual

site

-LRB-

Figure

-RRB-

Moreover

the

rate

constant

for

phosphodiester

hydrolysis

by

bound

to

the

immobilised

DNA

equals

that

from

the

fast

phase

of

the

reaction

of

on

the

one-site

plasmid

in

bulk

solution

-LRB-

Figure

-LRB-

b

-RRB-

-RRB-

Although

these

experiments

employed

different

types

of

DNA

substrates

in

one

case

an

oligoduplex

attached

to

a

coated

bead

and

in

the

other

case

a

plasmid

in

free

solution

they

still

support

the

view

that

the

fast

phase

from

the

reaction

of

on

the

one-site

plasmid

is

due

to

the

enzyme

bound

to

a

single

site

Discussion

The

tetrameric

SfiI

restriction

is

now

one

of

the

principal

experimental

systems

used

for

the

analysis

of

long-range

interactions

between

distant

DNA

sites

Two

of

its

subunits

-LRB-

a

primary

dimer

-RRB-

form

one

DNA-binding

cleft

and

the

other

two

form

a

second

cleft

on

the

opposite

side

of

the

protein

the

clefts

are

separated

by

about

Å

SfiI

recognises

a

palindromic

DNA

and

the

two

subunits

in

each

primary

dimer

interact

symmetrically

with

the

cognate

site

in

much

the

same

way

as

a

standard

dimeric

restriction

enzyme

with

a

single

DNA-binding

cleft

However

virtually

no

DNA

is

cleaved

by

the

complex

of

wt

SfiI

with

one

recognition

site

instead

almost

all

of

its

reactions

are

due

to

the

complex

with

two

sites

Moreover

it

has

to

be

the

scissile

site

in

both

clefts

a

complex

carrying

the

recognition

sequence

in

one

cleft

and

in

the

other

cleft

either

a

noncognate

sequence

bp

different

or

a

phosphorothioate

derivative

of

the

correct

sequence

fails

to

cleave

either

DNA

Hence

information

about

the

occupancy

of

each

DNA-binding

cleft

and

the

susceptibility

of

the

sequence

in

each

cleft

must

somehow

be

transmitted

through

the

protein

to

the

other

cleft

Å

away

The

transfer

of

information

between

the

two

DNA-binding

clefts

in

a

tetrameric

restriction

enzyme

must

occur

through

the

interface

between

the

two

primary

dimers

Inspection

of

the

crystal

structure

of

SfiI

bound

to

two

cognate

duplexes

suggests

that

may

play

a

pivotal

role

at

this

interface

-LRB-

Figure

-RRB-

The

aromatic

ring

of

this

tyrosine

sits

in

a

hydrophobic

pocket

in

the

opposite

subunit

and

its

hydroxyl

group

forms

an

inter-subunit

hydrogen

bond

across

the

dimer

interface

The

removal

of

the

hydroxyl

function

from

by

the

conservative

mutation

to

phenylalanine

resulted

in

a

protein

that

retains

the

tetrameric

structure

of

wt

SfiI

and

its

ability

to

bind

two

DNA

sites

at

the

same

time

to

cleave

two

sites

in

cis

concertedly

-LRB-

Figures

and

-RRB-

In

these

respects

is

like

wt

SfiI

though

its

turnover

rate

on

the

two-site

plasmid

is

about

lower

than

wt

on

account

of

its

slow

dissociation

from

the

doubly-cut

product

-LRB-

Figure

-LRB-

c

-RRB-

-RRB-

However

differs

markedly

from

wt

on

DNA

with

one

SfiI

site

-LRB-

Figure

-RRB-

The

wt

enzyme

acts

more

slowly

on

one-site

than

on

two-site

DNA

because

it

acts

on

the

one-site

DNA

in

trans

bridging

two

separate

molecules

of

the

DNA

and

on

the

two-site

DNA

in

cis

looping

out

the

intervening

DNA

In

contrast

the

steady-state

reaction

of

on

the

plasmid

with

one

SfiI

site

gave

biphasic

kinetics

a

fast

phase

that

was

considerably

faster

than

its

reaction

on

the

two-site

plasmid

but

which

then

declined

exponentially

to

a

slower

phase

whose

rate

matched

that

on

the

two-site

DNA

Both

phases

involve

multiple

turnovers

of

the

enzyme

Further

experiments

showed

that

the

fast

phase

is

due

to

acting

at

individual

sites

and

that

the

slower

phase

is

due

to

its

synaptic

complex

with

two

DNA

sites

-LRB-

Figures

and

-RRB-

can

thus

cleave

one-site

DNA

without

the

need

to

bridge

two

separate

molecules

of

the

DNA

-LRB-

Figure

-RRB-

The

net

effect

of

this

Tyr

Phe

mutation

is

thus

to

switch

SfiI

from

an

enzyme

showing

high

activity

at

two

sites

and

low

activity

at

one

site

to

the

exact

opposite

low

activity

at

two

sites

and

high

activity

at

one

site

It

inverts

the

ratio

of

its

turnover

rates

on

two-site

versus

one-site

substrates

from

for

wt

SfiI

to

approximately

for

-LRB-

as

measured

from

its

fast

phase

on

the

one-site

DNA

-RRB-

Kinetic

model

for

SfiI

cooperativity

Cooperative

action

by

an

oligomeric

protein

is

generally

accounted

for

by

either

the

Monod

Wyman

Changeux

-LRB-

MWC

-RRB-

or

the

Koshland

Nemethy

Filmer

-LRB-

KNF

-RRB-

scheme

In

both

schemes

each

subunit

of

the

oligomer

exists

in

either

an

inactive

low-affinity

tense

-LRB-

T

-RRB-

state

or

an

active

high-affinity

relaxed

-LRB-

R

-RRB-

state

In

the

following

the

terms

T

and

R

apply

to

a

primary

dimer

of

SfiI

a

single

DNA-binding

unit

so

that

the

tetramer

is

noted

as

T/T

T/R

or

R/R

-LRB-

Figure

-LRB-

a

-RRB-

-RRB-

the

subscript

S

will

denote

bound

substrate

In

the

MWC

model

the

T

R

transition

occurs

in

all

or

of

the

subunits

so

that

the

protein

never

contains

both

T

and

R

subunits

at

the

same

time

On

the

other

hand

the

KNF

model

proposes

sequential

T

R

transitions

upon

ligand

binding

to

each

subunit

allowing

hybrid

oligomers

containing

both

T

and

RS

subunits

-LSB-

To

keep

the

protein

active

the

reactions

reported

here

were

carried

out

by

adding

to

mixtures

of

enzyme

and

DNA

and

thus

might

appear

to

start

from

an

enzyme

DNA

complex

rather

than

from

free

enzyme

However

these

reactions

had

the

same

kinetics

as

those

started

by

adding

enzyme

to

the

DNA

mix

so

the

dissociation

of

any

DNA

bound

in

the

pre-equilibrium

must

be

rapid

compared

to

the

subsequent

steps

Hence

these

reactions

can

be

considered

as

starting

from

the

free

enzyme

In

the

absence

of

metal

ions

as

in

the

pre-equilibria

SfiI

binds

weakly

and

nonspecifically

to

DNA

-RSB-

The

properties

of

wt

SfiI

its

cooperative

binding

to

two

DNA

duplexes

and

its

enhanced

activity

on

two-site

over

one-site

substrates

can

be

accounted

for

readily

on

either

the

MWC

or

the

KNF

model

The

wt

enzyme

almost

always

needs

to

bind

two

copies

of

its

recognition

site

before

it

can

cleave

DNA

thus

on

DNA

with

one

site

it

acts

primarily

in

trans

bridging

two

DNA

molecules

The

wt

reaction

on

a

one-site

DNA

thus

proceeds

largely

via

the

RS/RS

state

to

the

virtual

exclusion

of

T/RS

or

R/RS

forms

On

a

DNA

with

two

sites

the

wt

enzyme

binds

both

sites

in

cis

to

again

yield

the

RS/RS

state

but

in

this

case

the

transition

from

the

free

T/T

form

to

the

doubly-liganded

RS/RS

structure

will

occur

much

more

readily

than

on

a

one-site

DNA

due

to

the

local

concentration

effect

for

sites

in

cis

In

contrast

the

mutant

of

SfiI

cleaves

DNA

readily

after

binding

just

one

site

so

that

a

substrate-induced

conformational

change

in

one

primary

dimer

to

give

the

hybrid

T/RS

state

is

sufficient

for

activity

The

presence

of

hybrid

species

is

allowed

by

the

KNF

scheme

but

not

by

the

MWC

scheme

thus

if

both

and

wt

SfiI

follow

the

same

mechanism

it

has

to

be

a

KNF

scheme

A

simplified

three-state

version

of

the

KNF

model

for

cooperative

action

by

a

dimer

as

opposed

to

the

full

nine-state

version

is

shown

in

Figure

-LRB-

a

-RRB-

in

this

scheme

the

substrate-binding

and

the

T

R

steps

always

occur

together

so

that

the

unliganded

T/R

and

R/R

states

the

partially

liganded

T/TS

and

R/RS

states

and

the

fully

liganded

TS/TS

and

TS/RS

forms

are

all

excluded

leaving

only

T/T

T/RS

and

RS/RS

It

also

coalesces

the

DNA-cleavage

and

product-release

steps

To

examine

whether

the

scheme

in

Figure

-LRB-

a

-RRB-

can

describe

the

reactions

of

both

wt

and

enzymes

on

both

one-site

and

two-site

substrates

the

differential

equations

for

the

change

in

concentration

of

each

species

with

time

were

solved

by

numerical

integration

using

a

range

of

different

values

for

each

of

the

six

rate

constants

and

k

for

the

first

DNA-binding

event

and

k

for

the

second

DNA-binding

event

and

and

for

DNA

cleavage/product

release

from

the

singly

and

doubly-liganded

protein

Numerous

trials

were

undertaken

with

different

values

until

a

match

between

model

and

experiment

had

been

obtained

The

values

used

for

and

for

wt

SfiI

differed

from

each

other

but

in

both

cases

the

same

sets

of

rate

constants

were

used

for

their

reactions

on

the

one-site

and

two-site

substrates

The

distinction

between

one

and

two

sites

was

modelled

instead

by

multiplying

-LSB-

S

-RSB-

the

association

rate

for

the

second

DNA

by

a

factor

Ω

to

account

for

the

local

concentration

of

one

DNA

site

in

the

vicinity

of

another

being

higher

for

sites

in

cis

than

for

sites

in

trans

by

definition

Ω

for

sites

in

trans

and

by

selection

Ω

for

sites

in

cis

Even

though

the

latter

value

was

selected

arbitrarily

it

reflects

experimental

and

large

-LRB-

-RRB-

variations

in

this

figure

made

essentially

no

difference

to

these

calculations

-LRB-

data

not

shown

-RRB-

For

both

enzymes

a

set

of

rate

constants

was

found

that

yielded

progress

curves

that

closely

matched

the

experimental

data

for

that

enzyme

on

both

one-site

and

two-site

substrates

-LRB-

Figure

-LRB-

c

-RRB-

-RRB-

for

wt

Figure

-LRB-

d

-RRB-

for

the

corresponding

experimental

data

are

shown

in

Figures

and

To

match

the

experimental

data

it

was

necessary

to

stipulate

that

both

rate

constants

leading

to

the

formation

of

T/RS

-LRB-

and

k

-RRB-

were

smaller

for

wt

SfiI

than

for

and

that

conversely

both

rate

constants

leading

away

from

T/RS

-LRB-

k

and

-RRB-

had

to

be

smaller

for

In

particular

for

had

to

be

held

at

a

low

value

comparable

to

that

for

the

DNA-cleavage/product-release

steps

from

the

T/RS

intermediate

-LRB-

-RRB-

in

order

to

simulate

the

biphasic

reaction

profile

on

the

one-site

DNA

-LRB-

Figure

-LRB-

b

-RRB-

-RRB-

-LSB-

If

was

set

either

much

smaller

or

much

larger

than

the

substrate

concentration

declined

linearly

with

time

In

addition

in

order

to

account

for

the

slower

turnover

rate

of

on

the

two-site

plasmid

had

to

be

fixed

at

a

lower

value

than

for

wt

SfiI

but

this

concurs

with

the

experimental

data

encompasses

both

DNA

cleavage

by

RS/RS

and

product

release

and

the

release

of

the

doubly-cut

product

is

slower

from

than

from

wt

SfiI

-LRB-

Figure

-LRB-

c

-RRB-

-RRB-

-RSB-

However

the

models

used

the

same

values

for

the

rate

constant

for

DNA

cleavage

by

the

complex

with

one

DNA

-LRB-

the

T/RS

state

-RRB-

The

fact

that

the

T/RS

state

leads

to

some

DNA

cleavage

by

but

virtually

by

wt

SfiI

is

due

to

the

differing

extents

to

which

this

state

is

populated

during

their

reactions

In

this

scheme

-LRB-

Figure

-LRB-

a

-RRB-

-RRB-

the

principal

effect

of

replacing

with

Phe

is

to

reduce

the

differences

in

free

energy

between

the

T/RS

state

and

either

the

T/T

or

the

RS/RS

structures

With

wt

SfiI

the

T/RS

state

is

very

strongly

disfavoured

relative

to

either

T/T

or

RS/RS

in

thermodynamic

terms

and

it

also

has

a

short

lifetime

so

that

wt

SfiI

cleaves

DNA

with

one

site

by

forming

the

RS/RS

complex

with

two

sites

in

trans

Even

when

a

high

value

is

allotted

to

the

wt

enzyme

for

-LRB-

the

rate

constant

for

DNA

cleavage

by

the

T/RS

state

-RRB-

virtually

all

of

the

DNA

is

still

cleaved

by

wt

via

its

RS/RS

state

In

contrast

the

T/RS

state

for

is

more

stable

thermodynamically

than

that

for

the

wt

enzyme

and

it

also

has

a

longer

lifetime

due

to

a

low

value

for

the

rate

constant

for

the

T/RS

RS/RS

step

Hence

with

the

one-site

DNA

is

cleaved

initially

via

the

T/RS

state

but

because

the

T/RS

RS/RS

transition

still

has

a

negative

ΔG

it

will

eventually

form

the

RS/RS

state

with

two

DNA

molecules

in

trans

Structural

model

for

SfiI

cooperativity

How

does

this

Tyr

Phe

change

stabilise

the

T/RS

intermediate

At

present

a

crystal

structure

is

available

for

wt

SfiI

bound

to

two

cognate

duplexes

but

not

for

the

free

protein

without

DNA

Given

the

scheme

in

Figure

-LRB-

a

-RRB-

the

solved

structure

is

presumably

akin

to

the

RS/RS

state

although

it

may

denote

a

precursor

to

the

catalytically

active

form

Nevertheless

the

cartoon

of

RS/RS

-LRB-

Figure

-LRB-

a

-RRB-

-RRB-

relates

to

the

known

crystal

structure

-LRB-

Figure

-LRB-

a

-RRB-

-RRB-

In

this

structure

the

two

subunits

that

make

up

the

primary

dimers

are

in

close

proximity

at

the

DNA-binding

surface

but

lie

distant

from

each

other

at

the

dimer

interface

In

almost

every

case

to

date

where

crystal

structures

are

available

for

a

restriction

enzyme

in

both

DNA-bound

and

free

forms

the

DNA-binding

cleft

has

an

open

configuration

in

the

absence

of

DNA

but

it

closes

up

around

the

DNA

in

the

complex

Hence

it

is

possible

that

the

two

DNA-binding

clefts

in

SfiI

also

have

open

configurations

in

the

absence

of

DNA

but

become

narrower

upon

binding

cognate

DNA

If

so

then

the

movement

of

two

subunits

towards

each

other

across

the

DNA-binding

cleft

might

result

given

rigid

body

motion

in

these

subunits

moving

away

from

each

other

at

the

interface

with

the

other

primary

dimer

-LRB-

as

illustrated

by

the

T/T

RS/RS

transition

in

Figure

-LRB-

a

-RRB-

-RRB-

The

interface

between

the

dimers

is

relatively

flat

and

is

almost

entirely

hydrophobic

in

nature

which

might

permit

each

subunit

to

slide

or

rotate

past

the

opposite

subunit

in

the

other

dimer

A

similar

scheme

for

intersubunit

motion

has

been

proposed

for

the

subunit

rotation

step

in

site-specific

recombination

by

γδ

resolvase

However

in

the

crystal

structure

of

wt

SfiI

bound

to

two

duplexes

the

hydroxyl

groups

of

in

all

four

subunits

make

direct

hydrogen

bonds

to

in

their

partner

subunits

across

the

dimer

interface

In

order

to

satisfy

the

hydrogen-bonding

potentials

of

both

and

these

interactions

are

likely

to

be

present

not

only

in

the

RS/RS

state

as

observed

in

the

crystal

structure

but

also

in

the

free

protein

the

T/T

state

even

though

the

free

protein

may

have

a

different

geometry

at

the

dimer

interface

On

the

other

hand

when

only

one

of

the

two

primary

dimers

changes

conformation

from

T

to

R

upon

DNA

binding

the

and

residues

in

the

liganded

RS

dimer

will

no

longer

be

in

register

with

those

in

the

unliganded

T

dimer

-LRB-

Figure

-LRB-

a

-RRB-

-RRB-

This

dislocation

may

destabilise

the

T/RS

state

as

neither

nor

will

have

a

suitably

positioned

hydrogen-bonding

partner

In

the

above

scheme

one

function

of

in

wt

SfiI

is

to

ensure

that

whenever

one

of

the

two

dimers

binds

DNA

and

undergoes

the

T-to-RS

transition

the

other

dimer

is

forced

to

follow

suit

and

to

undergo

immediately

its

own

switch

from

T

to

R

so

as

not

to

accumulate

the

highly

unstable

T/RS

state

However

without

the

interaction

as

in

the

mutant

the

T-to-RS

transition

in

one

dimer

would

no

longer

compel

the

other

dimer

to

undergo

straightaway

its

own

T-to-R

switch

The

hybrid

T/RS

form

is

more

stable

and

has

a

longer

lifetime

in

the

reaction

of

compared

to

wt

so

it

accumulates

sufficiently

and

exists

long

enough

to

allow

for

some

DNA

cleavage

events

while

in

this

state

Materials

and

Methods

Mutagenesis

Plasmids

containing

the

genes

coding

for

the

SfiI

restriction

endonuclease

pRRS-SfiIR

and

the

SfiI

modification

methyltransferase

were

provided

by

Ira

Schildkraut

-LRB-

New

England

Biolabs

-RRB-

and

were

used

to

transform

Escherichia

coli

first

with

and

then

with

pRRS-SfiIR

The

mixture

of

plasmids

isolated

from

this

strain

was

employed

for

site-directed

mutagenesis

of

the

SfiI

restriction

gene

by

the

QuikChange

method

-LRB-

Stratagene

-RRB-

The

resultant

PCR

products

were

used

to

transform

E.

coli

-LRB-

-RRB-

The

plasmids

were

isolated

from

the

transformants

and

the

derivatives

of

pRRS-SfiIR

were

sequenced

across

the

entire

gene

for

the

mutant

SfiI

endonuclease

-LRB-

University

of

Dundee

Sequencing

Service

-RRB-

only

the

mutation

had

been

introduced

Proteins

and

DNA

Wt

SfiI

and

were

purified

from

E.

coli

cells

that

had

been

transformed

successively

with

and

pRRS-SfiIR

-LRB-

or

a

derivative

-RRB-

as

described

previously

The

purified

enzymes

were

stored

at

°C

Concentrations

of

wt

SfiI

and

the

mutant

were

assessed

by

absorbance

at

nm

using

an

extinction

coefficient

of

M

cm

where

M

refers

to

the

tetramer

All

SfiI

molarities

are

thus

cited

for

the

tetramer

with

Mr

Protein

structures

were

analysed

with

INSIGHT

II

v.

-LRB-

Accelrys

San

Diego

-RRB-

and

surface

charge

was

evaluated

with

GRASP

v.

The

plasmids

and

which

contain

one

or

two

SfiI

sites

respectively

were

used

to

transform

E.

coli

and

the

resulting

transformants

were

grown

in

minimal

media

containing

MBq/l

-LSB-

-RSB-

thymidine

-LRB-

GE

Healthcare

-RRB-

The

covalently

closed

form

of

the

plasmid

was

purified

by

density

gradient

centrifugations

The

preparations

contained

mostly

the

SC

form

of

the

monomeric

plasmid

with

generally

as

either

dimer

or

OC

DNA

concentrations

were

assessed

by

absorbance

at

nm

All

oligonucleotides

were

obtained

from

Sigma

Genosys

as

HPLC-purified

samples

Oligonucleotides

were

annealed

to

give

the

duplexes

shown

in

Table

by

heating

a

mixture

of

two

oligonucleotides

with

complementary

sequences

to

°C

and

then

slowly

cooling

overnight

to

room

temperature

The

mixtures

generally

contained

more

of

the

bottom

strand

than

the

top

strand

to

ensure

that

all

of

the

top

strand

was

incorporated

into

the

duplex

The

bottom

strand

of

the

duplex

-LRB-

Table

-RRB-

was

phosphorylated

at

its

end

by

using

polynucleotide

kinase

-LRB-

Roche

-RRB-

and

-LSB-

-RSB-

ATP

-LRB-

GE

Healthcare

-RRB-

as

described

before

Analytical

ultracentrifugation

Sedimentation

to

equilibrium

was

done

at

°C

in

a

Beckman

XL-A

analytical

ultracentrifuge

using

an

rotor

with

six

channel

centrepieces

The

three

sample

channels

contained

μl

of

or

with

either

or

wt

SfiI

in

AUC

buffer

-LSB-

mM

Tris

HCl

mM

NaCl

mM

and

mM

dithiothreitol

-LRB-

DTT

-RRB-

pH

-RSB-

The

proteins

and

the

DNA

both

at

μM

had

been

dialysed

previously

against

AUC

buffer

and

the

reference

channels

contained

μl

of

the

dialysate

Centrifugation

was

carried

out

sequentially

at

and

rpm

After

and

h

at

each

speed

the

differences

in

absorbance

at

nm

between

samples

and

references

were

recorded

as

a

function

of

centrifugal

radius

-LRB-

r

-RRB-

At

all

speeds

the

profiles

at

and

h

were

identical

showing

that

equilibrium

had

been

reached

Centrifugation

was

then

continued

for

h

at

rpm

to

obtain

the

baseline

offset

For

each

sample

plots

of

against

centrifugal

radius

at

varied

speeds

were

fitted

globally

to

the

equation

for

a

single

homogenous

species

to

give

values

for

the

molecular

mass

-LRB-

M

given

here

as

Mr

values

-RRB-

Ar

and

are

the

absorbances

at

r

and

at

the

reference

respectively

ν

is

the

partial

specific

volume

ρ

is

the

buffer

density

ω

is

the

angular

velocity

and

B

is

the

baseline

offset

Values

for

ν

and

ρ

were

obtained

as

before

DNA-binding

studies

Binding

reactions

were

performed

by

adding

aliquots

of

wt

SfiI

or

in

dilution

buffer

to

either

or

or

to

mixtures

of

the

two

duplexes

to

give

solutions

that

had

in

μl

of

binding

buffer

nM

SfiI

tetramer

and

a

total

duplex

concentration

of

nM

Dilution

buffer

is

composed

of

mM

Tris

HCl

mM

β-mercaptoethanol

mM

EDTA

vol/vol

glycerol

mM

spermine

and

vol/vol

Triton

-LRB-

pH

-RRB-

Binding

buffer

is

composed

of

mM

Tris

HCl

mM

NaCl

mM

mM

β-mercaptoethanol

and

μg/ml

bovine

serum

albumin

-LRB-

pH

-RRB-

After

min

at

room

temperature

the

samples

were

mixed

with

μl

of

binding

buffer

containing

-LRB-

wt/vol

-RRB-

Ficoll

and

applied

to

polyacrylamide

gels

in

mM

Tris

borate

-LRB-

pH

-RRB-

and

mM

as

described

previously

After

electrophoresis

the

gels

were

scanned

in

a

Molecular

Dynamics

PhosphorImager

with

illumination

at

nm

Enzyme

assays

For

kinetic

experiments

the

final

reaction

mixtures

contained

nM

DNA

either

or

and

various

concentrations

of

either

wt

SfiI

or

the

mutant

in

μl

of

reaction

buffer

at

°C

Reaction

buffer

is

composed

of

mM

Tris

HCl

mM

NaCl

mM

mM

DTT

and

mg/ml

bovine

serum

albumin

-LRB-

pH

-RRB-

The

reactions

were

carried

out

by

adding

μl

of

-LRB-

mM

-RRB-

to

μl

of

enzyme

and

DNA

in

reaction

buffer

lacking

-LRB-

apart

from

some

trial

reactions

initiated

by

adding

enzyme

to

DNA

in

reaction

buffer

-RRB-

Aliquots

-LRB-

μl

-RRB-

were

removed

from

the

reactions

at

various

times

after

adding

-LRB-

one

was

removed

before

adding

to

serve

as

a

zero

time

point

-RRB-

and

vortexed

immediately

with

μl

of

an

EDTA

stop

mix

The

samples

were

analysed

by

electrophoresis

through

agarose

under

conditions

that

separate

the

various

products

from

each

other

and

from

the

SC

substrate

The

concentrations

of

each

form

at

each

time

point

were

determined

by

scintillation

counting

All

values

given

here

are

presented

as

the

means

from

three

independent

experiments

with

error

bars

to

denote

standard

errors

Zero-order

rates

were

evaluated

using

GRAFIT

-LRB-

Erithacus

Software

-RRB-

to

fit

the

initial

phases

of

substrate

utilisation

and/or

product

formation

to

linear

plots

For

some

reactions

-LRB-

viz.

Figure

-LRB-

b

-RRB-

-RRB-

the

concentration

of

DNA

substrate

was

fitted

to

an

initial

exponential

decline

followed

by

a

zero-order

phase

For

the

various

reaction

mechanisms

considered

here

-LRB-

Figure

-RRB-

extents

of

substrate

utilisation

were

modelled

by

solving

the

differential

equations

for

the

time-dependent

changes

in

the

concentrations

of

each

species

during

the

course

of

the

reaction

by

numerical

integration

in

BERKELEY

MADONNA

Some

reactions

on

also

contained

the

oligoduplex

These

typically

contained

in

μl

of

reaction

buffer

at

°C

nM

enzyme

-LRB-

either

or

wt

SfiI

-RRB-

nM

-LRB-

-RRB-

and

nM

samples

were

taken

at

varied

times

after

initiating

the

reaction

with

and

analysed

as

described

above

to

determine

the

amount

of

that

had

been

converted

to

its

linear

form

Immobilised

oligonucleotides

Streptavidin-coated

magnetic

beads

from

Promega

-LRB-

pmol

of

streptavidin

-RRB-

were

mixed

with

-LRB-

pmol

-RRB-

in

μl

of

SSC

washed

in

SSC

and

resuspended

in

μl

of

reaction

buffer

without

The

requisite

concentrations

of

either

wt

SfiI

or

the

mutant

were

then

added

before

initiating

the

reactions

with

-LRB-

final

concentration

mM

-RRB-

Aliquots

-LRB-

μl

-RRB-

were

taken

from

the

reactions

at

varied

times

and

quenched

with

loading

dye

-LRB-

mM

NaOH

mM

EDTA

formamide

bromophenol

blue

and

xylene

cyanol

-RRB-

After

incubating

first

at

°C

for

min

and

then

on

ice

for

min

the

samples

were

analysed

by

denaturing

gel

electrophoresis

through

a

polyacrylamide

gel

in

TBE

buffer

-LSB-

mM

Tris

borate

and

mM

EDTA

-LRB-

pH

-RRB-

-RSB-

at

V/cm

and

°C

The

gels

were

fixed

in

-LRB-

vol/vol

-RRB-

acetic

acid

and

-LRB-

vol/vol

-RRB-

methanol

dried

exposed

overnight

and

scanned

in

a

PhosphorImager

The

amounts

of

intact

and

cleaved

strands

of

the

duplex

were

quantified

using

ImageQuant

-LRB-

Molecular

Dynamics

-RRB-

and

the

decline

in

substrate

concentration

with

time

fitted

to

a

single

exponential

in

GRAFIT

auc, analytical ultracentrifugation
dtt, dithiothreitol
hex, hexachlorofluorescein
knf, koshland–nemethy–filmer
lin, linear
mr, relative molecular mass
mwc, monod–wyman–changeux
oc, open circle
sc, supercoiled
wt, wild type
allostery
dna–protein interaction
protein–protein interaction


CD24 Expression is an Independent Prognostic Marker in Cholangiocarcinoma CD24 has been described as an adverse prognostic marker in several malignancies . This study evaluates CD24 expression in cholangiocarcinoma and correlates the findings with clinicopathologic data and patient survival . Between 1996 and 2002 , 22 consecutive patients with cholangiocarcinoma were treated at our institution . Demographic data , SEER stage , pathologic data , treatment , expression of CD24 , mitogen-activated protein kinase -LRB- MAPK -RRB- , phosphorylated MAPK , and survival were analyzed . The majority of the tumors demonstrated CD24 -LRB- 81.8 % -RRB- and p-MAPK -LRB- 87 % -RRB- expression . A negative association was noted between the expression of CD24 and p-MAPK . Median survival for patients with low expression of CD24 was 36 months and high expression was 8 months . Median survival for patients who received chemotherapy with low CD24 expression was 163 months , and for seven patients with high CD24 expression , it was 17 months -LRB- p = 0.04 -RRB- . With the addition of radiation therapy , median survival for patients with low expression of CD24 was 52 months and high expression was 17 months -LRB- p = 0.08 -RRB- . On multivariate analysis , the use of chemotherapy -LRB- p = 0.0014 , hazard ratio 0.069 -RRB- and the CD24 overexpression -LRB- p = 0.02 , hazard ratio 7.528 -RRB- were predictive of survival . CD24 is commonly expressed in cholangiocarcinoma , and overexpression is predictive of poor survival and possibly of lack of response to chemotherapy and radiation therapy . These findings may improve selection of patients for the appropriate treatment modality and the development of CD24-targeted therapy . Introduction Malignancies of the biliary tract are uncommon in the Western world . Two-thirds arise in the gallbladder and the remainder in the biliary tree or periampullary region . Cholangiocarcinoma or bile duct cancer is a rare but lethal malignancy with an incidence of 1 -- 2 cases per 100,000 patients in the United States .1 Clinicopathologic factors predictive of survival include curative resection , tumor stage and grade , serum bilirubin level < 10 mg/dl , low CA19-9 level , hepatitis viral infection ,2 -- 4 lymphovascular or portal vein invasion ,5 intrahepatic satellite lesions , inraductal papillary compvonent , tumor angiogenesis ,6 and DNA ploidy .7 Reports of molecular markers predictive of survival in cholangiocarcinoma include cluster of differentiation CD24 ,8 MMP-2 , TIMP-2,9 cholinesterase level ,10 MUC-4,11 cyclin D1 ,12 VEGF-C ,13 p27 ,14 p53 , and p73 .15 Recently , CD24 has been described in a wide variety of malignancies and shown to be a prognostic marker in several solid tumors including colorectal , stomach , lung , prostate , ovarian , and breast .16 -- 21 CD24 is a small , heavily glycosylated , mucin-like , cell-surface protein expressed in developing cells including pre-B cells , keratinocytes , and renal tubular epithelium .22 -- 24 It functions as an alternative ligand of P-selectin , an adhesion receptor expressed on activated endothelial cells and platelets which can enhance the metastatic potential of CD24-expressing tumor cells .25 -- 28 CD24 has apoptotic activity , and its cross-linking induces the sustained activation of p38 MAPK -LRB- mitogen-activated protein kinases -RRB- -- the magnitude of which may determine the survival or death of pre-B cells .29 An improved understanding of the molecular pathways involved in the pathogenesis and progression of cholangiocarcinoma will contribute to the development of targeted therapy . This study correlates CD24 and MAPK expression with patient survival in cholangiocarcinoma with the objective of identifying a subset of patients who may benefit from targeted molecular therapy . Patients and Methods Clinical Data After obtaining approval of the Institutional Review Board , a review of the tumor registry at Roswell Park Cancer Institute identified 31 consecutive patients with histologically proven cholangiocarcinoma between 1996 and 2002 . Twenty-two patients had adequate tissue for further histopathologic studies and constitute the basis of this study . Medical records of these patients were reviewed for demographic data including age ; gender ; surveillance , epidemiology , and end results -LRB- SEER -RRB- stage at presentation ; treatment ; and survival from the time of diagnosis . Immunohistochemical Staining For most of the patients , diagnosis was established by examination of conventional hematoxylin and eosin -LRB- H&E -RRB- - stained slides and , in the remainder diagnosis , was confirmed with ancillary techniques including immunohistochemistry and special histochemistry with mucin and PAS stains . Uniform tissue fixation techniques were used for all patients . For each patient , a representative block containing adequate neoplastic and nonneoplastic tissue was selected . Five-micrometer tissue sections from these blocks were placed on charged slides and dried in a 60 °C oven for 1 h. Upon return to room temperature , the slides were deparaffinized in three changes of xylene and rehydrated using graded alcohols . Endogenous peroxidase was quenched with 3 % aqueous H2O2 for 15 min and washed with phosphate buffered saline with 0.05 % Tween-20 -LRB- PBS/T -RRB- . CD24 primary antibody was obtained from BD Biosciences -LRB- clone ML5 -RRB- and used with the recommended incubation time and antigen retrieval procedures . The primary antibody used for MAPK was obtained from Cell Signaling and for phosphorylated/activated MAPK from Santa Cruz . After a PBS/T wash , 0.03 % casein -LRB- in PBS/T -RRB- was used as a block for 30 min followed by the application of the primary antibody to the slides for an hour or overnight . Another PBS/T wash was followed by exposure to the biotinylated secondary antibody for 30 min . A third PBS/T wash was followed by exposure to the streptavidin -- peroxidase complex for 30 min . A PBS/T wash was followed by the application of the chromogen DAB -LRB- DAKO , Carpinteria CA , USA -RRB- for 5 min . The slides were then counterstained with hematoxylin , rinsed with water , dehydrated , and cleared , and a coverslip was placed . The use of biomarkers , related controls , and interpretation of results using the Histoscore system for quantification of results have been described previously by our group .30 Histoscore was defined as the product of the percentage of positive cells and the intensity of stain . The grade of positive staining depended upon the intensity of staining -LRB- 0 : no staining , 1 : weak , 2 : moderate , and 3 : strong staining -RRB- and the percentage of cells stained . The final score was calculated as a sum of each stain intensity multiplied by the percentage of stained cells in the area of interest . For example : if tumor showed 50 % weak , 30 % moderate , and 20 % strong staining , the score assigned was . Histological Grading The cholangiocarcinoma tissue specimens were also stained by routine H&E stains . The specimens were graded based on the degree of tumor differentiation using the World Health Organization -LRB- WHO -RRB- system . Statistical Analysis Association between biomarker expressions in tumor tissue was investigated using the Kendall 's tau . Biomarker expressions were classified as high and low based on whether their scores were above or below the median value , and survival between low and high expressions was compared using the log-rank test . The Kaplan -- Meier method was also used to estimate the survival curves and median survival . The Cox 's proportional hazards survival analysis was used in the multivariate analysis of survival data to explain the effect of biomarker expressions together with other diagnostic parameters . Patient demographics including age , tumor grade , SEER stage , and treatment received were considered as possible parameters for explanatory variables in the model . All statistical tests were two-sided with statistical significance level at 5 % . Results Patient Characteristics Of the 22 patients included in the study , 7 were males and 15 females . The median age was 66.5 years -LRB- range : 35 -- 77 -RRB- . SEER staging was local in 1 -LRB- 4.6 % -RRB- , regional in 14 -LRB- 63.6 % -RRB- , and distant in 7 -LRB- 31.8 % -RRB- patients . Differentiation of the tumor was classified as grade 1 in 3 -LRB- 13.6 % -RRB- , grade 2 in 9 -LRB- 40.9 % -RRB- , and grade 3 in 10 -LRB- 45.5 % -RRB- patients according to the WHO classification . Treatment for cholangiocarcinoma included surgery only -LRB- n = 8 -RRB- , surgery and chemotherapy -LRB- n = 5 -RRB- , surgery and radiation -LRB- n = 1 -RRB- , chemotherapy only -LRB- n = 2 -RRB- , and all three treatment modalities -LRB- n = 5 -RRB- , and one patient did not receive any treatment . Immunohistochemical Staining Normal bile duct staining was used to set the score intensity . Most of the bile ducts were negative . Occasionally , they demonstrated weak focal and incomplete staining as seen in Fig. 1 . The cholangiocarcinoma cells were scored as 1 + when they demonstrated weak expression of CD24 , 2 + for moderate expression , and 3 + for strong expression of CD24 . Figure 1 depicts cholangiocarcinoma positive for CD24 expression adjacent to normal biliary epithelium . The majority of the tumors demonstrated CD24 -LRB- 81.8 % -RRB- and p-MAPK -LRB- 87 % -RRB- expression . Immunohistochemical staining for these proteins was higher in malignant tissue in comparison to normal biliary epithelium . The pattern of staining was usually a combination of cytoplasmic and apical , and few specimens demonstrated the apical pattern only . Figure 1Moderately differentiated cholangiocarcinoma with overexpression of CD24 . Normal bile duct -LRB- right upper corner -RRB- demonstrated weak apical staining , whereas neoplastic cells had strong apical and cytoplasmic staining -LRB- 20 × -RRB- . Relationship Between Biomarkers A negative association was suggested between the expression of CD24 and phosphorylated/activated p-MAPK -LRB- Kendall 's τ = − 0.32408 , p = 0.0501 -RRB- . Survival Median survival was 36 months for nine patients with low expression of CD24 and 8 months for 13 patients with high expression of CD24 as shown in Fig. 2 . The median survival for five patients who received chemotherapy with low CD24 expression was 163 months , and for seven patients with high CD24 expression , it was 17 months -LRB- Fig. 3 , p = 0.04 -RRB- . Median survival for four patients treated with radiation in the presence of low CD24 expression was 52 months , and it was 17 months for two patients with overexpression of CD24 -LRB- Fig. 4 , p = 0.08 -RRB- . Overexpression of CD24 continued to affect survival adversely despite the overall improvement noted with the addition of radiation therapy . Multivariate analysis using the Cox 's proportional hazards survival analysis demonstrated that overexpression of CD24 -LRB- p = 0.02 , hazard ratio 7.528 -RRB- and use of chemotherapy -LRB- p = 0.0014 , hazard ratio 0.069 -RRB- were predictive of survival -LRB- Table 1 -RRB- . There was no significant association noted between survival and patient 's age , sex , SEER stage , grade of the tumor , surgery , radiation therapy , or expression of MAPK . Figure 2Kaplan -- Meier survival curve for patients of cholangiocarcinoma with low and high levels of CD24 expression -LRB- n = 22 -RRB- . p = 0.02 . Low CD24 -LRB- -- -- -- -- -- -- -- -RRB- , high CD24 -LRB- - - - - - - - - - - - - -RRB- . Figure 3Kaplan -- Meier survival curve for patients who received chemotherapy with low and high levels of CD24 expression -LRB- n = 12 -RRB- . p = 0.04 . Low CD24 -LRB- -- -- -- -- -- -- -- -RRB- , high CD24 -LRB- -- - -- - -- - -- -RRB- . Figure 4Kaplan -- Meier survival curve for patients who received radiation therapy with low and high levels of CD24 expression -LRB- n = 6 -RRB- . p = 0.08 . Low CD24 -LRB- -- -- -- -- -- -- -- -RRB- , high CD24 -LRB- -- - -- - -- - -- -RRB- . Table 1Prognostic Variables for Survival in 22 Patients with CholangiocarcinomaVariablenMedian survival -LRB- months -RRB- Survival -LRB- p value -RRB- Multivariate analysis -LRB- p value -RRB- Age < 681117 -LRB- 8 -- 36 -RRB- 0.60 > 681111 -LRB- 5 -- 52 -RRB- GenderMale712 -LRB- 8 -- 163 -RRB- 0.52 Female1517 -LRB- 6 -- 52 -RRB- Grade1 , 21215 -LRB- 6 -- 52 -RRB- 0.9931011 -LRB- 8 -- * -RRB- SEER stage1 , 21511 -LRB- 6 -- 52 -RRB- 0.483728 -LRB- 6 -- 163 -RRB- ChemotherapyNo106 -LRB- 4 -- 13 -RRB- 0.00050.0014 Yes1252 -LRB- 17 -- 163 -RRB- RadiationNo168 -LRB- 6 -- 28 -RRB- 0.12 Yes644 -LRB- 17 -- * -RRB- SurgeryNo38 -LRB- 0 -- 28 -RRB- 0.17 Yes1917 -LRB- 7 -- 52 -RRB- MAPKLow1017 -LRB- 8 -- 36 -RRB- 0.68 High1128 -LRB- 5 -- 163 -RRB- P-MAPKLow1013 -LRB- 8 -- 52 -RRB- 0.34 High1136 -LRB- 4 -- 163 -RRB- CD24 < 120936 -LRB- 13 -- 163 -RRB- 0.100.02 > 120138 -LRB- 6 -- 28 -RRB- * The estimate was not provided because the upper limit of the survival curve had not reached a 50 % failure rate.CD denotes cluster of differentiation.p-MAPK denotes phosphorylated form of mitogen-activated protein kinase . Discussion The physiologic function of CD24 is incompletely understood but it has been shown to increase tumor proliferation , cell adhesion , motility , invasion , and apoptosis .22 -- 24,31 Selectins are cell adhesion molecules involved in the rolling adhesion of leukocytes to endothelial cells and platelets under the shear forces of circulation , and P-selectin expressed by thrombin-activated platelets and endothelial cells is a major ligand for CD24 on carcinoma cells .26,27 This suggests that CD24-expressing tumor cells can disseminate more readily due to their capacity to form thrombi with activated platelets or to adhere to endothelial cells . Friederichs et al. 28 have demonstrated that the carbohydrate sialylLex abundantly expressed on human cancers is essential for CD24-mediated rolling of tumor cells on P-selectin , and in its absence , human adenocarcinoma cells failed to arrest and colonize the lungs . CD24 , a metastasis-associated protein , has been recently identified as a downstream target of Ral signaling .32 Ral GTPases are important mediators of transformation , tumorigenesis , and cancer progression . Microarray by immunohistochemistry of a human bladder cancer identified CD24 as a novel Ral-regulated target and a prognostic biological marker . In this study , 81.8 % of patients with cholangiocarcinoma expressed CD24 . Median survival for patients with overexpression of CD24 was significantly shorter , and the addition of chemotherapy improved survival . A negative association was noted between the expression of CD24 and p-MAPK . The use of chemotherapy in patients with low expression of CD24 was associated with a median survival of 163 months compared to 17 months in patients with a high CD24 expression -LRB- p = 0.04 -RRB- . The use of radiation therapy in patients with low expression of CD24 was also associated with an improved survival than with overexpression of CD24 although the data did not attain statistical significance possibly due to the small number of patients in this series . It has been reported by Taguchi et al. 29 that the cross-linking of CD24 induces apoptosis in Burkitt 's lymphoma enhanced by a B-cell antigen receptor -LRB- BCR -RRB- - mediated signal . They observed that simultaneous cross-linking of pre-BCR clearly inhibited CD24-mediated apoptosis in pre-B cells . CD24 cross-linking also induces the sustained activation of p38 MAPK , and whether pre-B cells survive or die may be determined by the magnitude of MAPK activation . Consistent with these observations , the present study suggests an inverse association between CD24 and p-MAPK , and eventual cellular proliferation or apoptosis might be a consequence of the dominant effect in a complex interplay of opposing influences .33 Our data indicate that high expression of CD24 remains an adverse prognosticator despite the use of additional therapy . Chemotherapy and radiation were noted to provide maximal survival benefit to low expressors of CD24 although the data for the use of radiation was statistically insignificant probably due to the small number of patients in this study . Furthermore , correlation between CD24 expression and radiation sensitivity has been noted to vary with the cell type as in human small cell lung cancer , and radiation doses required to induce apoptosis of CD24-negative human ALL -LRB- acute lymphoblastic leukemia -RRB- cells were higher than those required for CD24-positive cells , suggesting that lack of CD24 surface antigen expression is associated with intrinsic radiation resistance .34,35 Hypoxia is a characteristic feature of tumor cells due to the sustained proliferation which progressively results in an acidic , nutrient-deprived , and hypoxic tumor microenvironment . Tumor oxygenation has been identified as an independent prognostic variable for locoregional control and overall survival following definitive irradiation for squamous cell carcinoma of the head , neck , and uterine cervix .36,37 Recent reports have indicated decreased efficacy of chemotherapy under hypoxic conditions in several tumor types including pancreatic cancer and testicular tumors .38,39 Because treatment failure was a consequence of hypoxia , the authors recommend novel treatment strategies aimed at improving tumor oxygenation or enhancing the treatment sensitivity of hypoxic tumor cells . Aimed at identifying potential oxygen-dependent markers in vascular endothelial cells for therapeutic intervention in tumor angiogenesis , Scheurer et al. 40 performed a broad-range transcriptomic analysis of selected extracellular matrix protein gene expression levels in human umbilical cord vein endothelial cells exposed in vitro to hypoxia for different time periods . They noted several genes transcriptionally upregulated including CD24 at late times of exposure to hypoxia , indicating that it was a useful marker of hypoxic activation in vascular endothelial cells . In the present series , low expressors of CD24 demonstrated greater survival benefit from chemotherapy and radiation than the high expressors , suggesting that its expression may be a marker for tumor hypoxia and response to therapy . This finding that shows that patients with low expression of CD24 may benefit from chemotherapy or radiation is of importance because it has been previously reported that adjuvant or palliative radiation had no effect on survival in patients with cholangiocarcinoma .41 However , the small number of patients in the present series limits interpretation of data suggesting that CD24 overexpression may be predictive of lack of response to radiation or chemotherapy . CD24 has been shown to be a prognostic marker for shortened survival and disease progression in several malignancies including colorectal , stomach , lung , prostate , ovarian , and breast cancers .16 -- 21 Weichert et al. report that in colorectal cancer , only the subset of patients with exceptionally strong cytoplasmic CD24 staining comprising 10 % of their study group demonstrated a markedly shortened mean survival of 31.5 months compared to 67.5 months for the remaining patients .16 They also reported that cytoplasmic CD24 staining pattern is prognostically more significant than the membranous pattern -- the biological significance of which was unclear . Su et al. noted a 51 % expression of CD24 by immunohistochemistry in intrahepatic cholangiocarcinoma as compared to the 81.8 % in the present series . They reported CD24 expression and tumor stage as independently predictive of survival on multivariate analysis and suggested membrane-bound CD24 protein as a potential target for immunotherapy .8 In conclusion , overexpression of the molecular marker CD24 in cholangiocarcinoma is predictive of poor survival . CD24 overexpressors demonstrated a lack of response to chemotherapy and possibly radiation therapy although these observations were limited by the small sample size . Additional properties of tumor proliferation , invasion , metastasis , and apoptosis make CD24 a potent target for specifically directed molecular therapy and its overexpression a potential criterion in the selection of patients for the appropriate conventional treatment modality .

B I O O O O O O B O O O O O O O O O O O O O O O O B I O B O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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cd24 expression
cholangiocarcinoma
survival