Reversibility of nucleosome conformation perturbed by urea

Vol.

85,

No.

December

4, 1978

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

29,1978

REVERSIBILITY M. The

OF

D.

Zamo,+

University and

Received

NUCLEOSOME

October

CONFORMATION

E. Olins,

PERTURBED

E. Wilkinson-Singley,

of Tennessee-Oak Biology Division, Oak Ridge,

and

A.

1446-1452

BY UREA*

L.

Olins

Ridge Graduate School of Biomedical Oak Ridge National Laboratory, Tennessee 37830, USA

Sciences

30, 1978

SUMMARY - Monomer nucleosomes (v, ) from chicken erythrocyte nuclei were diluted into and urea was removed by dialysis. 9 M urea plus 0.2 mM EDTA (pH 7.0), The y thus obtained were fractionated by sucrose gradient ultracentrifugation. Each fraction was examined in 0.2 mM EDTA for reversibility of v, structure perturbed by urea. At least to urea was restored to the original structure, as 30% of the initial amount of v exposed shown by sedimentation veloci ‘cr , electron microscopy, circular dichroism, thermal melting, and fluorescence of v1 labeled with -N&(3-pyrene) maleimide on thIol greups of H3 histone. As an approach somes the

which

effects

in our

might

laboratory

(17),

nuclease

digestion

of chromatin most

(21,

studies,

Abbreviations:

solvents,

disruptive

ionic effects

(8-l

l),

22)

It has been

are hand,

reversibility

reversed our

which

18),

recent

in v, exposed

studies

nucleosomes;

NPM,

*Research sponsored by the Division of Biomedical Department of Energy, under contmct W-7405-eng-26 by NIH grants to DE0 (GM 19334), and by Grant t This research was performed while Dr. Zama was Radiological Sciences, Chiba-shi, Japan.

Government's right to The U.S. license in and to the copyright purposes, is acknowledged.

have

shown

0 M urea.

1446

structure

the

have

been

X-ray

(17,

19,

20),

conformational

and

changes

concentration

employed

14). that

more

In the

-N-(3-pyrene)

extensive

present

denatur-

studies

we

have

by 9 M urea.

maleimide.

and

Environmental with the Union NSF 21498 to ALO.

on leave

of

previously

low-angle

urea

perturbed

retain covering

0006-291X/78/0854-1446$01.00/0 Copyright 0 1978 by Academic Press, Inc. AN rights of reproduction in any form reserved.

(8,

studies

made

microscopic

highest

of urea

conformation

pH were

(12-16),

that

of nucleo-

extensive

on chromatin

suggested

(4)

to 7-l

and

electron

is the

by removal

of nucleosome

monomer

15,

or transitions

(l-3),

strength,

spectroscopic

(14,

studies.

states

chromatin

of urea

denaturation

occurs

“1 ’

conformational

active

by 4 or 5 M urea,

the other

the

The

thermal

of v1 structure

examined

organic

of hydrodynamic

induced

of the On

ation

or other

by means

of the

in transcriptionally

(4-7).

scattering

for

occur

of urea

observed

to identification

from

the

Research, Carbide

National

a nonexclusive this paper,

U.S. Corporation,

Institute

of

royalty-free for governtiental

Vol.

85,

No.

MATERIALS

BIOCHEMICAL

4, 1978

AND

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

METHODS

previously at -20°C solutions

Monomer nucleosomes from chicken erythrocytes were The KC&soluble nucleosomes described (23, 24). -5O), then diluted in 0.2 mM EDTA, pH 7.0 (A260 just before use.

obtained employed with the

by use of methods were stored frozen oppropriate buffer

0.2 mM measured molority

Stock solutions of urea (-10 M, ultrapure grade, Schwarz/Mann) were made in Freshly made solutions exhibited pH 7.0. Each urea solution EDTA (pH 7.0). refmctometrically against 0.2 mM EDTA at the same temperature, and urea was calculated from the analytical formula described elsewhere (4).

was

The removal of urea from nucleosome solutions was done as follows: Dialysis tubing containing 2 ml of w (diluted into 9 M urea, 0.2 mM EDTA, pH 7.0) was put in 40 ml of 9 M urea, 0.2 mM EbTA (pH 7.0), 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 mM dithiothreitol . The dialysis ogainst decreasing urea concentrations was done at 4”C, with continual stirring, by a dropwise addition (-10 ml/hr) of 0.2 mM EDTA solution containing 0.1 mM phenylmethylsuifonyl fluoride and 0.1 mM dithiothreitol to the outer solution down to the final urea concentration (-2.5 M), followed by an extensive dialysis against 0.2 mM EDTA. The same procedure was applied to urea-denatured v, labeled with NPM. The preparation of NPM-v, complexes and their fluorescence and other properties have been presented in our previous papers (5, 6). with a Beckman Model E Sedimentation experiments of renatured v, were performed analytical ultracentrifuge equipped with scanner optics. Scanner data were recorded at 265 nm. Sucrose (0.5%) was added to half of each solution to stabilize against mechanical and thermal mixing. Sedimentation coefficients were determined at 30,000 rpm in an An-G rotor. The data were recorded at 22°C and corrected to S20 values with standard errors *W as indicated in Results. Electron microscopy and thermal melting experiments described previously (4). Circular dichroism measurements J-40A spectropolarimeter at room tempemture. The molar of DNA (E = 6,500) was used in calculations of the molar of nucleo&es, C 01,.

recording wavelength

Fluorescence of NPM-y complexes spectrofluorometer by the procedure was at 340 nm.

was measured described

Sucrose

sedimentation

were performed by the methods were performed on a Jasco extinction coefficient at 260 nm ellipticity, expressed per mole

in a Perkin-Elmer elsewhere (5, 6).

MPF-44 The excitation

RESULTS

nucleosomes urea

-

After

was dialyzed

on preparative urea-treated lo),

out linear

v

1’

while

sedimentation Y

were from

the

with

urea

medium. gradients.

where

the

a considerable

It was calculated

and

mixed

sucrose

u1 sediment

(fraction control

gmdient

The

y

native

proportion that

to a final

In Fig. control

about

30%

1447

velocity

of urea-treated

concentration

of 9 M urea,

th us obtained

in 0.2

1 it is shown

that

v,

have

mM

certain

a sedimentation

EDTA

the

were

fractions

run of

peak

of urea-treated

v, sediments

more

of urea-treated

v, sediment

above

slowly fraction

thon 10.

Vol.

85,

No.

BIOCHEMICAL

4, 1978

O

AND

5

10

15 FRACTION

TCIP

Figure v .

1. Preparative sucrose (A) Renatured urea-treated respectively. (8) Native lb, linear sucrose gradient containiig rotor (Beckman) at 35,000 rpm

Sedimentation sucrose

RESEARCH

20

25

COMMUNICATIONS

30 BOTTOM

NO.

gradient ultmcentrifugation of urea-treated v, and v The arrows denote the positions for fractions 1’ v . Each sample in 0.2 mM EDTA was loaded on a 0.2 mM EDTA (pH 7.0). Centrifugation was in and 4°C for 12 hr.

coefficients

gradients.

BIOPHYSICAL

were

for

the

obtained

renatured

for

two

fractions

urea-treated

v,

(10

and

(fraction

6) from

native 6 and 5-20% an SW 41

the

10) was

9.01

f

0.14;

between

the

two

for

urea-treated

v,

or unfolding

of

SO,W

the

value

is 9.31

particles

6,

for

untreated

within

the

other

hand,

on the

v, .

This

experimental was

difference

in

uncertainties.

6.52

f 0.06,

S20,w The

which

value

suggests

swelling

particle. SDS-gel

normal

electrophoresis

amounts

relative

of the

deficiency

no evidence normal

histones

We

renatumtion

cutoff

remains

gradient

H4,

seen

particles

Middle

weight

H2A,

the of some

H2B)

in fractions H4.

of urea-treated

in fractions

6 and

We

7.

attempted

Station,

6,000-8,CQO);

understand

fractions

New

however,

basis

of the

of H3 and

tubing

which yields

loss of H3 and the

a

provided yield

has a smaller approximately

H4 during

the

wall

of

(Spectrum

were

inside

showed

1, whereas,

a better

dialysis

the

v,

Electrophoresis

York),

H4 onto

8-l

to obtain

by use of Spectropor

Village

Adsorption

step.

(H3,

loss of H3 and

Inc.,

do not yet

sucrose

H4 was

urea-treated

(molecular

tubing

inner

of proteolytic

Industries,

same.

of the

of H3 and

sedimenting

Medical size

0.11

is probably

in fraction the

f

pore the

dialysis

of the dialysis

a possibility.

Electron

microscopy

-

The

exposed

urea-treated

v

observations

on the

1’

v,

disruptive

Figure

effects

2 presents

the

to 9 M urea of urea

electron

resemble on the

1448

short

ultmstructure

microscopy rods

(Fig.

of control 2D).

of v , have

and

Similar been

Figure 2. Dark-field electron microscopy (B) rwnatured urea-treated v1 (fraction lo), (D) v, in 9 M urea. All samples contained

of urea-treated v, . (A) (C) renatured urea-treated 0.2 mM EDTA (pH 7.0).

Native v1

vl, (fraction

6),

. ..

.,“,

Vol.

85,

No.

BIOCHEMICAL

4, 1978

I 220

I 260

1

I

WAVELENGTH

Figure

3.

y ((fraction

-

AND

BIOPHYSICAL

I 300

RESEARCH

%-k---j

(nm)

COMMUNICATIONS

15

FRACTION

NO.

Circular dichroism of urea-treated u . (A) Chicken DNA (-- -), native renatured urea-treated u, (fracdon 10) ( -), renatured urea-treated VI C81p: - -), v1 in 9 M urea (-----). molecular ellipticity per mole of All samples contained 0.2 mM EDTA. (B) o -, Ellipticity of renatured nucleotides. urea-treated LIP as a function of the fraction number from sucrose gradient ultracentrifugation (seeFig. 1); --0 --, the relative fluorescence intensity at 450 nm of w, labeled with NPM as a function of fraction number. Excitation wavelength was at 340 nm. -1, 6) (-

previously

reported

fraction

10 appears

unfolded

v,

3A.

becomes identical of our ticities

values indicates treatment

-

The

28 shows

that

after

urea

is dialyzed

out,

v, , whereas

fraction

6 consisted

of partially

dichroism

spectra

of urea-treated

v,

and v,

ellipticities

the with

previously

The

spectra exhibit

recovery

as a function

and

the

the

urea-treated

v,

within

a-helix number

increasing

fraction

DNA

subsequent

are

similar

structure dialysis.

1450

the

and

(260-300 (fraction

(i.e.,

of the

shown

in

nm) 10) yield uncertainties

molecular

sucrose

number; to the

are

a-helix

experimental

content

of fraction

10 and

dominant

spectrum

nm,

higher

the

suppressed

renatured

with

by fraction

9 M urea

and

slightly

of both

in 9 M urea

at 260300

decreased

reached

(4),

lost

DNA.

dichroism

are

circular

is completely

of naked

of urea-treated

plateau

of normal

reported

nm)

measurements,

The

after

that

circular

tation.

fact

dichroism

(~240 like

Fig.

2C).

As has been

contribution

However,

20).

to consist

(Fig.

Circular Fig.

(4,

gradient

at 281

and

values

of control

the secondary

structure

ellipsedimen223 v, .

nm This

of histones

Vol.

85,

No.

BIOCHEMICAL

4, 1978

AND

BIOPHYSICAL

TEMPERATURE

RESEARCH

COMMUNICATIONS

(“C)

Figure 4. Thermal denaturation at 260 nm of urea-treated v, . Derivative melting profiles are shown. All samples contained 0.2 mM EDTA (pH 7.0). Native VI (-), ureatreated v1 (fraction IO) (-), urea-treated v1 (fraction 6) (-----).

Fluorescence structure urea

of NPM-v,

urea-treated in

The

in contrast teristic

Fig.

of the

fraction

complexes

3B,

to the

mM

at -75”C,

very Melting

v ; the main 1 some difference transition

v,

melt

with

urea-treated

transition

v,

premelt

but

region.

considerably

data

exhibit v,

10)

is slightly

there

is slightly

The

urea-treated

compared

EDTA

higher

(fraction

(5,

plots

of the

and

a main

was used from

thermal transition

as solvent

that

of native and

6) exhibit

with

native

v, .

30%

of the

initial

6).

thermally

hyperchromicity

v,

charac-

treatment

different

1A).

above

VI were

at 59°C mM

(Fig.

fluorescence

urea

the

fluorescence,

particles

after

0.25

v,

excimer

any

the

centered

(fraction

reduced

9 exhibit

the derivative

in which

removed of the

urea-treated

urea-treated

premelt

then

ultracentrifugation

structure

4 shows

or quaternary

to 9 M urea,

that

renatured

a wide

published

is at 75”C,

in the

suggests

Figure

7.0).

of the

do not

and

(pH

to the

8 which

quaternary

tertiary

of the

fraction

Native

EDTA

to that

above

observation

and/or -

similar

at 67”C,

fraction

This

tertiary

Native

similar

complexes

below

denaturation

denaturations.

(25).

NPM-v,

their

in 0.2

was very

excimer.

9 maintain

denatured

the

complexes

NPM

Thermal

the

v

NPM-VI

As shown

To investigate

weexposed NPM-v complexes 1’ 1 fractionation profile by sucrose gradient

of urea-treated

by dialysis.

-

complexes

also

a main

DISCUSSION The

present

study

demonstrates

to 9 M urea

restore

their

native

compact

demonstrated

that

Several tin

studies

treated

studies does

with

(4) not

have

we

up to 5 M urea, have

dissociate

shown histones

that from

that

at low treatment DNA.

at least

structure the

after

histones

ionic

remain

strength

of u , with It is not

1451

ureo

(8, at least

yet

clear

amount

is removed associated

12,14,

why

exposed

by dialysis. with

26).

8 M urea

of v,

DNA In our

plus

we obtain

0.2

in chromaprevious mM

less than

EDTA com-

Vol.

85,

plete

No.

recovery

investigators

of native-like to chemically

renatured

velocity

BIOCHEMICAL

4, 1978

modified

particles.

Acknowledgment experiments

and

ul. modify

- We thank in the data

AND

Nonetheless,

the

urea-unfolded

Dr. A. analysis.

BIOPHYSICAL

v,

P. Butler

RESEARCH

procedures and

obtain

for

described significant

his help

in the

COMMUNICATIONS

here

will

amounts

permit of

sedimentation

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1452

67,

E. B., and (P. 0. P. Nucleic