31P NMR of DNA in eukaryotic chromosomal complexes

Vol. 102, No. 3,198l October

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 885-890

15, 1981

j’P J,A.

NMR OF DNA IN EUKARYOTIC CHROMOSOMAL COMPLEXES DiVerdi*,

S.J.

Opella*,

R.-I,

N.R.

Ma+,

Kallenbach+

Departments of Chemistry* and Biology+ University of Pennsylvania Philadelphia, Pennsylvania 19104 and N.C. State

Received

August

24,

Seeman

Department of Biology University of New York Albany, New York

1981

DNA complexed with histones in solyple chromatin and with protamines in t.he heads of sperm has been studied by P NMR spectroscopy. Because of the large size of these nucleoprotein structures, methods of high resolution solid state NMR were employed. Proton decoupled 31 P NMR spectra of these complexes in solution yield anisotropic chemical shift powder patterns, which indicate that the DNA is substantially immobilized by interactions with the proteins. Rapid rotation of these samples at the magic angle gives single line spectra with an isotropic chemical shift indistinguishable from DNA in the absence of proteins or that in mononucleosome core particles; this argues that packaging of the DNA by the proteins does not introduce ma,ior distortions in a predominant fraction of the phosphodiester linkages present. INTRODUCTION The DNA within the

polymer

free

eukaryotic in solution;

greater

it

compaction

is

contribute

to the condensation

which

cell

than

factor

a flattened center,

(2,3).

cylinder

with

on a string”

(core

+ linker) structures

enormously

subunit

Several

levels

about

length units

unit

the

contains

145 base pairs of linker have

in electron

of coiling

structure

only

been visualized

a core

first

with

of

a

resembling

around

the histone

The next

to

to DNA in the

particle

as the well (4).

the

of DNA by about

of DNA wrapped

DNA complexed

micrographs

of linear

of histones

length

compared

the extent

chromatin,

The binding reduces

condensed

that

of DNA in nuclear

The nucleosome

and a variable

Repeated

(1).

in detail.

mononucleosomes

of five

is

has been estimated

103-fold

has been characterized

fundamental

nuclei

known level

a protein H1.

“beads of folding

0006-291X/81/190885-06$01.00/0 885

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BIOCHEMICAL

Vol. 102, No. 3,198l

involves

formation of helical

the linker fibrils

AND

BIOPHYSICAL

fibrils

from several nucleosomes connected by

DNAsegments and histone Hl (4-6).

ultimately Few details

RESEARCH COMMUNICATIONS

Further coiling

gives rise to the extremely compact nuclear material of the organization

component, protamine, packages

the DNAin the extremely small sperm heads; this protein acids, of which 24 are arginine and 6 cysteine (8,g). cysteines form intermolecular The two nucleoprotein the higher folding

cross-links

Apparently

the

amongthe protamines (10).

DNA. The soluble chromatin prepared

nuclei by mild nuclease digestion consists of about

ten nucleosomes linked together; here, these fibrils

has only 47 amino

systems studied here thus represent extremes of

levels of eukaryotic

from chicken erythrocyte

(1,7).

of DNAand proteins in bull sperm

chromatin are known. A single major protein

level of folding

of these

in the low ionic strength conditions used

have an apparent diameter of 1Onm(4-6).

Only the first

beyond that of the nucleosome core particle

is present in

the soluble chromatin.

In contrast,

at least two orders of magnitude more in

linear compaction of the DNA is present in demembranatedheads of bull sperm. Previous NMRstudies of chromosomalcomplexes have been concerned with mononucleosomecore particles

(11-13).

Larger and potentially

complexes have not been studied because their average out the dipolar and chemical shielding sible for the overwhelming linewidth NMR, radiofrequency

irradiations

more interesting

slow reorientation interactions

rates do not

that are respon-

of resonances in solids.

In solid state

and mechanical sample spinning substitute

for molecular motions as line narrowing mechanisms. The usefulness of this approach for the study of biological

supramolecular structures

has been

demonstrated for viruses (14-16) and high molecular weight duplex DNA (17).

ANDMETHODS Chicken erythrocyte chromatin in solution was prepared by lysing red blood cells with Nonidet NP-40 detergent (Sigma). The resulting nuclei were briefly digested with microccocal nuclease (Worthington) according to Lutter (19). After stopping the reaction with the addition of EDTAthe material was dialyzed into 1OmMtris buffer with 1mMpH7 and pelleted by spinning at 40,000

886

Vol. 102, No. 3,198l

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rpm for 18 hours in a Ti50 rotor. The final NMR sample had an OD of 650 at 260nm in a volume of approximately 0.3ml. The DNA of the chromatin sample was analyzed by electrophoresis on a 0.5% agarose + 2.5% polyacrylamide gel as described in reference (12) after being released from the chromatin by treatment with proteinase-K and sodium dodecyl sulfate. The DNA in the soluble chromatin is approximately 2300 daltons. Therefore, there are about 12 nucleosome units per particle. Polyacrylamide gel electrophoresis of the chromatin proteins showed that histones Hl, H2a, and H4 were present. H2b’ H3' A suspension of bull sperm was demembranated by treatment with Nonidet detergent. The sperm tails were removed by sonication and the final samples were concentrated by low speed centrifugation. The duplex DNA samples were high molecular weight material from cell thymus. The sample preparation is described in reference (17). MR spectra were obtained on a homebuilt double resonance spectrometer with 31 P resonance frequency of 60.9 MHz (14,171.

EWJLTS

AND DISCUSSION

The lineshapes

of the

chemical

anisotropy,

shift

remove

the

spectrum

influence

the

tensor;

this

rigid

lattice

values

is

of motional

averaging;

while

faster

those

DNA in solution set

average heads

the

11.8

slower

reduce

Figure

in solution

The asymmetric

shift give

motions

powder

pattern

for

since

compared

to 11.8

kHz are damped out

observed

Aa for

30% from

the

source

the

particle

means that

the DNA-protein

static

value

of the reduction or local

except

structural

will

width

not and alter

relatively

fast chromatin

bending

in solution It

fluctuations

887

it

spectrum,

near

completely sperm

1B and 1C. and is

a

are

rapid

interactions. is

is difficult

to say that

motions

of DNA that

by the protein-DNA

1A.

the

times

Ao =132ppm

motions

detection

and bull

in Figures

chromatin

for

the lineshape.

these

shown

pattern shielding

affect

correlation

Soluble

the

In a magnetic

rotational

complexes

of Figure

chemical

with

soluble

result,

powder

the timescale

this

NMR spectra

striking

the

it

that

1A is

of the

Au q 194ppm.

that

by was used to

Figure

determines

anisotropy.

the 3’P

decoupling

interactions,

the observed

1D shows

chemical

proton

breadth

kHz which

motions will

power

of the phosphodiester

to a total

has backbone

(17);

high

1 are determined

The discontinuities

DNA.

this

of Figure

dipolar

fibrous

corresponds

of 3.5T

10-6

since

of 3’P-‘H

of solid

reflect

field

3’ P NMR spectra

reduced to fully

The by about characterize

must be from motions

of limited

amplitude

that

of are

BIOCHEMICAL

Vol. 102, No. 3,19Bl

AND

BIOPHYSICAL

02

RESEARCH COMMUNICATIONS

I 3 I ’ I ( I 8 200

D

PPM

-200

Figure 1. 31P NMR spectra of DNA and nucleoprotein complexes. A) Solid 9) Chicken erythrocyte chromatin in fibrous calf thymus DNA (Sigma). C) Bull sperm heads in solution. D) High molecular weight solution. calf thymus DNA (Miles) in solution. Spectra A, 9, and C resulted from cross-polarization with 1 msec mix time and 1 set recycle delay. Data was acquired for 10 msec with a Z.jmT ‘H decoupling field. Spectrum D was from n/2 pulses rather than cross-polarization. Figure 2. A) Soluble chromatin in solution. sample in an Andrew type rotor spinning at with respect to the applied magnetic field

fast

compared

to the

tude,

such as rotation

would

be that

spectra

from

ment;

ths

static

about

the

techniques

are

sub,iected

polarization, isotropic believe

were

‘H spins

obtained

in order

relies

31 P- ’ H dipolar

for

the 31 P NMR spectra shift this

position liquid-like

and because

it

histone

Hl give

varies as samples similar

older.

888

This

pulses

means

chromatin instead

resonance

on the powder

chromatin we do not

that

samples

the

pattern.

We

fraction

present from

of

of cross-

near

a minor

activity

31 P NMR spectra,

The

pattern.

the existence

When soluble

Since

spectrum

of the experi-

mechanism.

intensity

ampli-

of the 31P

namely

represents

of large

of the

sensitivity

effect,

nuclease

in relative get

shape powder

a narrow

component

material

without

state

exhibit

of the endogenous

in size

the

n/2 radiofreauency

DNA in view

were

by cross-polarization

(0 ppm) superimposed spectral

the

symmetric

spins.

degraded

and increases

then

as a transfer

solid-like

to nonselective

such motions

to increase

on a solid

couplings

selects

axis,

of an axially

lA-1C

procedure

the

a single

Same as figure 1B. B) Chromatin kHz at the magic angle ( 8 =54.7) a 25 mscc acquisition time.

If

kHz timescale.

characteristic

in Figures

spins

11.8

2.5 with

sample

in this to sample

samples think

of

with

the narrow

and

Vol. 102, No. 3,198l

resonances ments

BIOCHEMICAL

represent

do not Magic

value.

rigorously

angle

position

observed

solution.

This

evidence

centerband centerband is

state

molecular

weight

that

subtle

narrow

centerband

there

or severe

clearly

make it

detectable

by 31P chemical profound.

of DNA by quite

compaction

results

can be gained strengths

in

by using

to vary

the

bull

is

not

separated

from

narrowing

of the stationary

to study

The picture

shift

spin

sensitive

the

emerging are

backbone

levels

of the DNA.

interactions,

is

associated

and filamentous

immobilization

nuclei,

that

measurements

at various

high

interactions

has a flexible

proteins

substantial

timescale

no

in mind

drastic

possible

sperm heads,

different

other

with

changes

of DNA by protein

DNA alone

chromatin,

in

of the

be borne

lineshape

complexes.

or not

packaging

the

particles

distortion

and the

ic

shift

core

side-bands

isotrop

chromatin

chemical

to structural

that

perturbations

in soluble

2 for

of environment

must

frequency

structural

However,

experi-

anisotropy.

NMR techniques

are

it

of spinning

while

effects

at the

geometrical

shifts

further shift

in Figure

type

However,

DNA and DNA-protein

dynamical

present

to its

mononucleosome

is a single

kHz spinning

demonstrate

the

anisotropy

shown

or isolated

THe presence

due to chemical

Solid

are

linkages.

by the 2.5

chemical

procedure

of 31 P chemical (18).

of DNA although

averages

modification

sensitivity

samples

that

RESEARCH COMMUNICATIONS

out.

DNA alone

of chemical

established

of the

is

for

of chemical

the

the

a single

implies

phosphodiester

fully

is

this

this

BIOPHYSICAL

regions

spinning

from

there

linker

rule

sample

The results

in solution;

that

mobile

AND

(17). viruses

the

of folding Greater

and magnetic

and

detail field

to motion.

ACKNOWLEDGEMENTS This

research

Society

(NP-2251,

National

Science

Starter

Research

is

a fellow

is

being

supported

the National

by grants

Institute

of Health

Foundation

(PCM-7705598,

Grant

the March

of the

from

A. P. Sloan

from

889

(CM-24266,

PcM-8004043),

of Dimes Birth

Foundation

the American

(1980-1982).

Cancer

CM-26467),

and a Basil Defect

Foundation.

the

O'Connor S.J.O.

Vol. 102, No. 3,198l

1. 2. 3. 4. 5.

;: 9.

10.

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and Kay, D.