Circular dichroism spectra and ethidium bromide binding of 5-deoxybromouridine-substituted chromatin

VoL64,No.

BIOCHEMICAL

1,1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

CIRCULAR DICHROISM SPECTRA AND ETHIDIUM BROMIDE BINDING OF 5-DEOXYBROMOURIDINESUBSTITUTED Claudio

Nicolini*

CHROMATIN

and Renato

Baserga**

Department of Physiology and Biophysics* and Department of Pathology and Fels Research Institute** Temple University Health Sciences Center 'Philadelphia, Pennsylvania 19140

Received

March

14,1975 SUMMARY

Changes in CD spectra of 5-deoxybromouridine (brUdRib)-substituted chromatin depend on the extent of thymidine replacement by brUdRib.With 20 and 14 per cent replacement, a blue shift and a marked increase in positive ellipticity are reported in the CD spectra of brUdRib-substituted chromatin, while with 4.9 per cent BrUdRib-substitution does not affect the replacement little effect is noticed. number of ethidium bromide primary binding sites of chromatin. It

has recently

moiety

by 5-deoxybromouridine

dichroism (1)

been reported

(CD) spectra

reported

a blue

(1,2)

(brUdRib)

of both shift

and a marked

had been

replaced

and Seale

substituted

(-45

more evident ported

per cent)

by brUdRib.

In both

and Bekhor

bromide

A possible et al., Bekhor chromatin

(1) (3)

who found

of brUdRib-substituted

a 4 - 15 per

ethidium

(3),

cent

in brUdRib-substituted

and Simpson on the other,

from

in the number

ascites

for

cells,

thymidine

obtained

by

the

brUdRib

Opposite

results

in positive

of primary

effect

was

were

re-

ellipticity

these binding

authors sites

for

chromatin.

(2)

may be found tumor

were

In addition,

the discrepancy

and Seale

at

had been extensively

a decrease

chromatin.

decrease

explanation

DNA.

et al.,

in which

thymidine

in protein-free

in circular

ellipticity

results

cases,

thymidine

Augenlicht

3T6 cells,

Similar in which

than

changes

Thus,

from

by brUdRib.

of the

in positive

HeLa cells,

by Lapeyre

found

dramatic

(2) with

in chromatin

of CD spectra

causes

increase

of DNA and chromatin

Simpson

replacement

DNA and chromatin.

260 nm in CD spectra 20 per cent

that

between

on one side, in the

in which

fact

the

results

and those that

the

of Augenlicht

of Lapeyre latter

the maximum extent

and

authors of thymidine

used

Vol. 64, No. 1, 1975

BIOCHEMICAL

replacement

by brUdRib

indeed,

changes

the

in CD spectra

ment by brUdRib. affect

In addition,

chromatin

measured

was 6.7 per

primary

by sensitive

AND BIOPHYSICAL

cent.

The present

depend it

communication

upon the extent

shows that

binding

RESEARCH COMMUNICATIONS

sites

for

of thymidine

brUdRib-substitution ethidium

spectropolarimetric

shows that, replace-

does not

bromide

when the

latter

are

methods.

MATERIALS AND METHODS The methodology for

the

isolation

for

ively, moieties

cent,

were

Model

Circular

J-40

Seale

but

(2)

were

Ethidium methodology (8).

This

optically

with

bromide

binding

assumes

active. correct

the number

ethidium

bromide

cent,

that

gave,

con-

respect-

of thymidine

CD spectra

described

for

reported

was measured

light

(1).

scattering

by Simpson

and

chromatin.

(6), only

by circular

Williams

assumption

(12),

so that

(7)

(in either

optical

sites

(7-11)

to be analytically

spectropolarimetric

sites

the and Baserga

acquire

secondary

has been proved the

using

and Nicolini

dye molecules so-called

dye molecules,

This

dichroism,

et al.,

intercalated

binding

extent

Three

in 10 mM Tris

the results

was measured

of primary

(1,4,5).

as previously the

medium,

of the

replacement

of chromatin

(correcting

sheared

that

bound

papers

spectropolarimeter,

et al.,

weakly

quantitatively

and 4.9 per

be noted

obtained

method while

to give

should

the determination

72 HIM, 36 vM and 7.2 PM, which

chromatin

of Dalgleish

activity, not

it

in brUdRib-containing

in previous

dichroism

recording

We have used unsheared artifacts),

used,

14 per cent

by brUdRib.

in a Jasco

and for

have been given

of brUdRib

20 per

of 3T6 cells

of DNA and chromatin

of brUdRib-substitution centrations

the growth

method

are

and

can be used

DNA or chromatin)

for

(8). RESULTS

Fig.1

shows that

substituted thymidine

chromatin moieties.

the

are dependent Under

in CD spectra

described

occurred

at brUdRib

only

changes

our

in CD spectra

upon the extent

experimental

by Augenlicht concentrations

between

(1)

of the

the dramatic

and by Simpson

of 72 and 36 PM.

190

nm of brUdRib-

of replacement

conditions,

et al.,

250-300

changes

and Seale

At 7.2 vM (4.9

(2) per

Vol.64,

Figure

cent

BIOCHEMICAL

No. 1, 1975

1.

Circular dichroism spectra between 300 and 250 nm of chromatin and of brUdRibfrom untreated 3T6 mouse fibroblasts ( -) substituted 3T6 chromatin at final brUdRib concentrations of 7.2 PM (-----), 36 PM (-.-a-.-) and 72 UM (. . . . *) in growth medium.

substitution)

the

as unsubstituted proteins

is

control

most

Fig.3

chromatin Both

bromide

saturation

the ellipticity of the

from curves

CD spectra

between

in Fig.4.

No difference

or brUdRib-replaced

of the

ratio is

cells are

300-350

shifted

200-255

nm, where

concentration

added

are

the

of brUdRib

the same for

grown

only

dye/DNA

20-25

phosphate.

per

cent

detectable

that

same as that used

to a maximum of 20 per

191

cent.

chromatin cent

of and for

DNA.

complexes

either

com-

replacement).

of protein-free bromide

of

(Fig.2). bromide

(20 per

among chromatins,

of

The amount

unsubstituted

in 72 FM brUdRib

same shape

the contribution

of chromatin

nm of chromatin-ethidium is

but has the

at 308 nm of chromatin-ethidium

intercalated

isolated

slightly

the CD spectra

regardless

shows

is

Between

important

as a function

ethidium

CD spectrum

chromatin.

chromatin,

plexes

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

are

unsubstituted

The shown

VoL64,No.

Figure

BIOCHEMICAL

1, 1975

2.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Circular dichroism spectra, between 200 and 255 nm, of chromatin from untreated 3T6 mouse fibroblasts ), and brUdRib(substituted 3T6 chromatin at final concentrations of brUdRib of 7.2 uM (-----) and 72 UM (. . . . .) in growth medium. The solvent was 0.01 M Tris-HCl, pH8.

DISCUSSION The results ation

for

Simpson other

side.

(2)

on

of Lapeyre

one side,

above the

only

a modest

shift

4.9 per

without

(Fig.1).

Lapeyre

and Bekhor

stitution

because‘they

were only

findings

of Lapeyre

the extent

cent

substitution

could

injecting

in a modest

increase

brUdRib incorporation

192

and

and Bekhor

(3)

on the

in shape

replacement.

of replacement

in positive not

(1)

of brUdRib

which,

an explan-

--et al.

and the change

on the extent (3)

seem to offer

of Augenlicht

ellipticity

an increase (3)

communication

and those

in positive

and Bekhor

slightly

results

the

are dependent

only

that

present

between

The increase

of chromatin

experiment

ology

in the

the discrepancy and Seale

spectra

reported

was 6.7

in our

in experimental of brUdRib

In the per

cent,

laboratory,

ellipticity the extent

of CD

gave

of CD spectra of brUdRib animals, into

sub-

a method-

DNA, unless

BIOCHEMICAL

Vol. 64, No. 1,1975

AND BIOPHYSICAL

R (added

Figure

3.

special

per cent

are

(Selma

by Lapeyre cation.

A possible

Lapeyre

and Bekhor

the

intercalation

more accurate

Silagi,

to obtain

personal

to explain

(3)

and the

explanation (3),

are

the

and measures

brUdRib-substitution

with

the

that

only

lack

could

discrepancy

be found

ethidium

of brUdRib

has several

ethidium

sites

communi by

(i.e.

different method

(12).

bromide

of reported

used

pitfalls

not qualitatively

and RNA polymerase

193

effect

in the methodology

binding

affect

the

in the present

the spectropolarimetric

bromide

of

by chromatin,

reported

that

primary

effect

between

bromide

of effect

but

while

does not

the well-known

of ethidium

quantitatively (15)),

a brUdRib-substitution

communication).

spectrophotometry,

only

sites

that

report

Interestingly,

on the binding

and Bekhor

sites

(13-14). in order

is more difficult

brUdRib-substitution

secondary

used

is necessary

on differentiation It

dye/DNA)

Ethidium bromide ellipticity at 308 nm as a function of the ratio of added dye/DNA (8)308 of ethidium bromide complexed with calf , chromatin from untreated 3T6 mouse fibroblasts thymus DNA &(M) and brUd f? ib-substituted 3T6 chromatin at final concentrations of brUdRib 7.2 PM (a---D), and 72 IIM (. . . . +) in growth medium. CD spectra were obtained in 0.01 M Tris-HCl pH8. Ellipticity is expressed in arbitrary units, i.e. millidegree per optical density of DNA (see text).

techniques

15-30

RESEARCH COMMUNICATIONS

from (7,8)

Our present binding,

compete

the

is finding

together for

the same

BIOCHEMICAL

Vol.64,No.1,1975

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

“Ill

Figure

binding

4.

Circular dichroism spectra of ethidium bromide bound to chromatin from untreated 3T6 mouse fibroblasts () and brUdRib-substituted 3T6 chromatin at final concentrations of brUdRib of 7.2 PM (-----) and72PM(. . . . .) in growth medium. The dye/DNA ratio was always 0.25 f 0.01. CD spectra were obtained in 0.01 M Tris-HCl pH8. The ellipticity is expressed in decimoles of ethidium bromide.

sites

stitution

(16),

are

does not

affect

in agreement the

with

number

previous

of chromatin

findings

that

binding

sites

brUdRib for

sub-

RNA polymerase

(5). ACKNOWLEDGEMENT This National

work

was supported

Cancer

by grants

CA12923

and CA08373

from

the

USPHS,

Institute.

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Augenlicht, Comm. 59,

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2.

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R.T.,

3.

Lapeyre,

J.,

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Hill, B.T., 71, 455-459.

Tsuboi,

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Hill,

and Baserga,

B.T.,

C.,

and Seale, and Bekhor, A.,

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R.L. I.

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1975

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