The binding of actinomycin to crab dAT; the nature of the DNA binding site

BIOCHEMICAL

Vol. 26, No. 2, 1967

THE

BINDING THE

OF ACTINOMYCIN

NATURE

Richard Gates Norman

Received

site

on DNA

of Cancer

omycin

per

pothesis

Several

are

examined.

for

actinomycin

base

The two-stranded

crab pairs

synthetic

et.

, 1965;

alternate

DNA

(Goldberg,

G is the critical

xc

sites.

residue

sites

et al.,

the sequence

Contribution

No.

the component

as crab

dAT,

which

binding

most

nucleosides

varying

1965).

Thus,

either

around

a G in order

3452

116

at about there for

the results,

only

suggest

binding

1965).

pair

that

1961).

7 base further

to occur,

Gellert, must

be

G rather However,

75 to 25%,

1 per

is some

in native

, 1962;

a CC base

from

bind-

1962 ; Cavalieri

c+&.

(Kersten,

constant

strong

and Felsenfeld,

1963)

for binding

the hy-

with

to occur

(Goldberg,

and Hurwitz,

at one actin-

for

and Reich,

at all

a GG content

is fairly

appear

the ratio

excludes

consistent

Neville,

with

saturates

probably

are

AMD

Smith,

for

is required

Rabinowitz,

not bind

Studies

with

for

of the binding

that with

which

sites

Kahan,

of DNA’s

(Gellert, for

does

Kahan,

than

result

the nature

We find

on one strand

Gellert,

dAT

binding

SITE

Davidson

with

the strong

hypotheses,

binding

in these

known

This

strong

present

a number

DNA

is 1:28,

pairs.

1964;

Since

deals

D (AMD).

a GpG sequence

and Nemchin.

BINDING

and Norman

communication

56 base

ing.

DNA

dAT;

29, 1966

present

that

of AMD

W. Hyman

antenarrius

of CC to AT

OF THE

TO CRAB

and Crellin Laboratories of Chemistry* and W. Church Laboratory of Chemical Biology California Institute of Technology Pasadena, California 91109

November The

AND BIOPHYSICAL RESEARCH COhVWNlCATlONS

in

the number pairs

requirement or repulsive

Vol. 26, No. 2, 1967

interactions

(either

the DNA

base

Crab

pairs.

fully

form,

nativ’e

veloped

in this

that

interactions

interest

(1963)

distortion

limit

estimate

of

the amount that

of actinomycin

an alternating

A method

for

the direct

would

DNA,

are

occurs

extend

less

likely

to investigate

low

the binding

with

of this

Widholm,

GC base

in a very

copolymer,

of the GC base

(Davidson,

In this

AT

the preparation

disruption

Laboratory

GpG

chains

is mainly

without

1965).

sequence

actinomycins

and Reich

peptide

by local

for

pairs.

and Wang, steric:

Fuller,

dAT

of GC base

or indirectly

neighboring

of the cyclic

three

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

steric

between

Hamilton,

effect

about

directly

structure)

of binding. steric

BIOCHEMICAL

material

pairs,

Nandi,

pairs

are

has been

de-

Olivera,

sufficiently

rare

so

furthermore,

It therefore

of actinomycin

3%

in its

Jensen,

to be important;

frequency.

about

the

seemed

to native

crab

of

dAT.

Experimental sancer of Smith

(1963)

component buoyant ‘nating are

antenarrius except

called

dAT

(2. 7% CC).

AMD

complex

washed The

studies

solution.

SW39 ml

also

1o-3

M - phosphate,

rpm

for

six hours

tube

contained loat 20°C

et al.

(1965).

pairs

(Widholm,

of the better

Cancer

were

made

by sedimenting

pH 7. pelleting

117

rotor

and Dohme. the DNA-

remaining known

in

amounts

in a carefully

two ml

Salt The

AMD

containing

with

borealis

Sharp,

of free

placed

is an alterits properties

Merck,

20% sucrose.

thereby

1965);

known

were

sulfate

DNA

from

and covered

&I NaCl,

This

a gift

and AMD

The

by the mercury-cesium

ml of solution

of DNA

by the method

out in the cold.

the concentration Three

polyallomer

essentially

carried

separated

D was

and measuring

prepared

were

to those

binding

determined)

bottom

was

Actinomycin

the supernatant (spectrally

steps

3. 5% GC base

similar

Equilibrium

was

of Davidson,

with very

all

dAT

method

copolymer generally

that

crab

density

DNA

of mineral

concentrations was

the AMD-DNA

oil. were

spun

at 42,000

complex.

The

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 26, No. 2, 1967

polyallomer

tubes

were

dripped

two ml of supernatant then

measured

solution

readily tubes

adsorbs

using

to glass

Plots dAT

shown

surfaces

1.

I

The

I

vertical

I

e-

.4

I

.(I AMDf

low

of free

free

a higher plot

nucleotides; The

AMD.

actinomycin

curves

concentration.

of these

data

in the strong

These

values For

was

lengths.

handled

with

poly-

and one site agree crab

well dAT,

per

with

I

f,

DNA

and

is the ratio

of

I

2.4

2.1

1 coordinate

a strong

binding

region

binding

118

reaction

uptake analysis

gives

14 (* 1) nucleotides, reported

is the concen-

binding

by further

A least-squares

the intrinsic

thymus

I I /a

followed

those

calf

CobfNt~ymus0 _

indicate

concentration

AMD

liter.

#

the horizontal

free

mole-’

concentration

coordinate,

1.2 I.6 2.0 x 106 mole liter-’

Fig. to total

to native

Crab dAT i I : ef’ I : A

10 -

tration

and was

and

and Discussion

‘2 -

AMD

AMD

1 and 5 cm path

of actinomycin

in Fig.

I.

bound

free

the bottom,

and pipets.

of the binding

are

one ml from

The

collected.

Results

crab

the side,

by spectrophotometry,

Actinomycin propylene

from

of AMD

at

of a Scatahard

K = 2. 3 (*. 5) X lo6 for

calf

thymus

by Gel1ert.s. constant

at

is

DNA.

(1965). 2. 5 (k.

5) x

106

Vol. 26, No. 2, 1967

-1

mole

liter,

56 base

and there

pairs.

Since

tude

as for calf

dAT

appears

one such

CC) have

site

for

data

neighbor

determined

are:

ApA

DNA’s,

binding

two

CC base

order

There

pairs for

by Schwartz,

on crab

is approximately

Cancer

= 0.0126;

of magnisite

in crab

Trautner,

TpT

or one per

the binding

site.

frequencies

= 0.0127,

nucleotides,

is of the same

typical

strong every

112 (i4)

constant

and other

nearest

been

per

the binding

thymus

binding

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is one site

to be a typical

The

These

BIOCHEMICAL

dAT.

borealis

dAT

and Kornberg CpA

= 0.0100,

(2.7%

(1962). TpG

=

0.0089;

GpA

= 0.0042,

TpC

= 0.0015;

CpT

= 0.0004,

ApG

= 0.0018;

0.0081;

ApC

= 0.0069;

GpG

= 0.0009,

CpC

= 0.0009;

TpA

= 0. 504; ApT

0.429;

CpG

residues

GpC

a’re followed

a non-G base

= 0.0007;

residue.

G residues these

by non-G;

If there

preceding

From

for

is no correlation

a G residue

Cancer

borealis

if all the binding

sites

that

on the same

adjacent

G’s

these

data,

0. 927 of the G residues

a G and the probability

do not have

data

= 0.0015.

of a base on either

dAT

involve

between

apply

a G residue, strand

are

are

needed

by

the probability

of a

a G, 0.87

exclude

of the

neighbor.

antennarius

to define

=

preceded

side as a nearest

the data

=

0. 939 of the G

following

to Cancer

GpT

If

dAT,

and

the possibility a strong

binding

site. Professor to us.

The

Don Crothers

nearest

0. 64 of the G’s are ceded tides

neighbor followed

prec.eding

frequencies

and following

a purine

as a nearest

neighbor.

consists

of a G plus

an adjacent

of one site

per .Apart

about

assuming

purine

argument

Cancer

show

borealis

and 0. 74 of the G’s no correlation

the G, l-(0. Thus,

out the following

64-O.

that

are pre-

between

the nucleo-

74) = 0. 52 of the G’s have

the assumption is consistent

that a binding with

site

the observation

two G’s. from

the distribution

is random,

for

by a pyrimidine

Thus,

by a pyrimidine.

has pointed

the nearest of CC base

and if E = frequency

neighbor pairs

frequencies, along

of occurrence

119

the chain. of an AT

nothing

is known

If the distribution base

pair,

the

BIOCHEMICAL

Vol. 26, No. 2, 1967

probability pairs

that

a sequence

followed is(n)

are

to or shorter

equal

= l/(1

the sequences steric

following

are

-E).

interaction

between

that

only

of them

the CC base

that

dAT

chain

but there

is sufficient

bunching

ber

of sites

to l/2.

Alternatively,

there

around

must

have

a G; for

example,

a neighboring We are

are

are

plies

ment

pairs

pair

grateful

to Dr. dAT.

J. Widholm

the U. S. P. H. S.

GM

for

is an additional

that

large

strong

a

distance.

a binding

at random

to decrease

= 28, half

unlikely this

a

binding

along

site, im-

the crab

the fractional

num-

sequence

require-

by Crothers

that the G

purine.

GM-10991 Grant

available

of such

the sequences

is intrinsically

the one suggested

of crab

under

half

if (2)

extend

(s - 1)AT

length

It seems

not distributed

the preparation from

that Thus,

would

CC base

l/2

- E).

pairs.

actinomycins

if every

Therefore, the observation

19 base

consistsof

Th e average

of 2 such

is In 2/(1

than

CC pair

- E).

The value

than11

longer

a given

is E n-1(1

by a CC pair

sequence

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

This

research

for

advice

has been

and instruction supported

on

by Grant

One of us (R. W. H. ) is an N. I. H.

Trainee

1262.

References Cavalieri, Davidson, Gellert, Goldberg, Hamilton, Hurwitz, Kahan, Kersten, Schwartz, Smith, Widholm,

L. and Nemchin, R. (1964). Biochim. Biophys. Acta, 87, 641. N., Widholm, J., Nandi, U.S., Jensen, R., Olivera, B.M., and Wang, J. C. (1965). Proc. Natl. Acad. Sci. , U. S. , 53, 111. M. , Smith, C. E., Neville, D. , and Felsenfeld, G. (1965F J. Mol. Biol., 11, 445, I. H., RabiGwitz, M. , and Reich, E. (1962). Proc. Natl. Acad. Sci., U.S., 48, 2094. L. D. , Fuller, W., and Reich, E. (1963). Nature, 198, 538. J., Furth, J. J., Malamy, M., and Alexander, M. (1962). Proc. Natl. Acad. Sci., U.S., 3, 1222. E., Kahan, F. M. , and Hurwitz, J. (1963). J. Biol. Chem., 238, 2491. W. (1961). Biochim. Biophys. Acta, 47, 610. M. N., Trautner, T. A., and Kornberg, A. (1962). J, Biol. Chem., 237, 1961. M. (1963).xochem. Biophys. Res. Comm., 10, 67. J. (1965). Ph.D. Thesis, California Institute of Technology.

120