Activation of the template activity of isolated rat liver nuclei for DNA synthesis and its inhibition by NAD

BIOCHEMICAL

Vol. 53, No. 2,1973

ACTIVATION

AND BIOPHYSICAL

OF THE TEMPLClTE ACTIVITY OF ISOLATED RAT LIVER NUCLEI FOR DNA SYNTHESIS AND ITS INHIBITION BY NAD L. Burzio*

Biomedical University,

Received

RESEARCH COMMUNICATIONS

and S.S.

Koide+

Division, The Population Council, New York, New York 10021

June 5,

The Rockefeller

1973

SUMMARY: The template activity of isolated rat liver nuclei for DNA synthesis assayed with E. coli DNA polymerase was found to be dependent upon the presence of Ca2+ Or p in the incubation medium. DNA was prepared from isolated nuclei subjected to conditions which activated the template and centrifuged in an alkaline sucrose gradient. The distribution profile showed that smaller fragments were formed, suggesting enhancement of endonucleolytic activity. When isolated nuclei were incubated with NAD to induce poly(adenosine diphosphate ribose) formation and were subjected to the activation conditions, the template for DNA synthesis remained unchanged. The distribution profile in an alkaline sucrose gradient of DNA prepared from these nuclei and control nuclei The present findings suggest that the template-activating syswas identical. tem for DNA synthesis was blocked when isolated nuclei were treated with NAD in vitro. Treatment

of isolated

rat

liver

nuclei

with

inhibition

NAD --in vitro resulted in an into DNA (1,2). The block

of &he amount of [3H]TTP incorporated was related to ADP* -ribosylation of nuclear proteins to be due to a suppression of the template activity nuclei the

or assay

chromatin

(2).

condition

currently

as measured tion

and that

activity the

with

(5).

inhibition

associated

In the

exogenous

process

present

of DNA synthesis with

a block

the

of these

studies

template

of isolated

DNA polymerase

the activation In the

course

used,

of the

was gradually

was associated

report

evidence

exerted

(3,4) and was demonstrated for DNA synthesis of

will

we found activated

with

activation

MATERIALS

that

under

liver

nuclei

on incuba-

an endonucleolytic

be presented

by poly(ADP-Ribose)

template

rat

to show that

formation

is

process.

AND METHODS

Materials. DNA polymerase of about 5000 units per mg of protein obtained from --E. coli was purchased from Biopolymers, Pine Brook, N.J. Preparation of Nuclei. Rat liver nuclei was isolated in a medium containing 0.25 M sucrose, 2 mM CaC12 (g/5 ml) as described in a previous report pellet was suspended in 2.3 M sucrose, 2 ti CaC12 (1) * The crude nuclear as described by Chauveau et al. (6). The suspension was centrifuged at The pellet of purified nuclei was suspended in 0.25 40,000 x g for 60 min. * Present address: Instituto de Bioquimica, Universidad Austral de Chile, Casilla 567, Valdivia, Chile. + To whom all correspondence should be addressed. ADP, adenosine diphosphate; EGTA, ethylene glycol his * Abbreviatton used: (2-aminoethylether)-N-N' tetraacetic acid.

Copyright 0 1973 by Academic Press. Inc. AN rights qf reproduction ill arzy form reserwd

572

BIOCHEMICAL

Vol. 53, No. 2, 1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

M sucrose, 2 mM CaC12. In some preparations the pellets were washed twice with 0.34 M sucrose and suspended in the same solution. Enzymatic Studies. Poly(ADP-Ribose) synthetase activity was assayed as described in a previous report (1). The template activity for DNA synthesis of isolated rat liver nuclei was assayed in a medium containing 75 mM TrisHCl (pH 7.4), 5 r&l MgC12, 2-mercaptoethanol, 0.1 mM each of dATP, dCTP, dGTP, 0.5 mM r3H]TTP (50 to 100 $i/mole), one unit of --E. coli DNA polymerase and nuclei containing about 25 pg of DNA in a total volume of 0.5 ml. The reaction mixture was incubated at 25' for 15 min. To study the effect of bivalent cations on the activation of the template, isolated nuclei were washed twice and resuspended in 0.34 M sucrose. The incubation medium contained 75 mM Tris-HCl (pH 7.4), 2 mM 2-mercaptoethanol, 150 mM sucrose, 0.25 mM EDTA, varied concentrations of bivalent cations, nuclei containing about 200 N of DNA in a total volume of 0.5 ml. EGTA was added to chelate endogenous Ca2+ in the nuclei preparation. The reaction mixture was incubated at 37O. At the designated time intervals aliquots of 50 ~1 were removed and assayed for template activity. To study the effect of NAD on the template activity, the isolated nuclei preparations containing 200 pg of DNA were incubated in a medium containing 75 mM Tris-HCl (pH 7.4), 5 mM MgC12, 2 mM 2-mercaptoethanol, 4 mM NAD, in a The mixture was incubated at 25O for 30 min. The unfinal vol of 0.5 ml. treated-control (without NAD) and the NAD-treated nuclei samples were washed twice with a solution of 0.25 M sucrose, 2 mM CaC12. Template activity of the nuclei preparations was measured before and after being subjected to the activating process. The incorporation of [14ClNAD or t3H]TTP was measured in a Packard Tri-Carb Liquid Scintillation Spectrometer, Model 3375. The material precipitated with 5% trichloroacetic acid was collected on a glass fiber filter (Reeve Angel, Clifton, N.J.) and processed as described in a previous report (1). Preparation of nuclei containing about 300 ng Alkaline Sucrose Gradient. of DNA was incubated in a medium containing E. coli DNA polytnerase which was used to assay for DNA synthesis. After incubation two vol of ethanol at -20' was added to the reaction mixture. The mixture was centrifuged and the pellet was washed once with cold 66% ethanol, dried under vacuum and suspended in 0.5 ml of a medium composed of 0.2 N NaOH and 5 mM EDTA. At this step the amount of radioactivity in the material precipitated with trichloroacetic acid was greater than 90%. A sample of 0.3 to 0.4 ml was layered on a 12 ml solution of 5 to 20% linear sucrose gradient containing 0.2 N NaOH, 0.8 M NaCl, 1 mM EDTA (7). The tubes were centrifuged in a Spinco Model L2-65 ultracentrifuge using a SW-40 rotor at 30,000 rpm for 17 hr at 5'. About 30 fractions of 15 drops each were collected from the bottom of the tube. Aliquots of 100 ~1 of each fraction were removed and the radioactivity in the trichloroacetic acid-precipitated material was measured as described by Bollum (8). DNA in the remainder of the fraction was determined by a modified method (9) of Burton (10). Other procedures. DNA was prepared according to Mannur (11). Protein was determined as described by Lowry et al. (12). RESULTS In the synthesis crease sis

for

activity nuclei

course

of control in activity

nuclei with

the activation was carried was very

of these

low.

studies

it

the duration of the

out

was observed

preparation

at 37'

However,

(without of the

template, (Fig.

incubation.

a time 1).

on incubation

573

that

the template

NAD) showed course

The template

To determine study

for

a gradual of the

activity

at 37O in a medium

DNA

inthe ba-

template

of fresh containing

Vol. 53, No. 2, 1973

BIOCHEMICAL

Preincubatian

time

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(min)

0

;b 0

1:

5

IO

Fraction

mM nicotinamide

Figure 1. Activation of template for DNA synthesis of control nuclei following incubation in a medium containing Mg2+ plus and NAD-treated nuclei were incubated at 3?to activate the cribed in the text. Aliquots were removed at the designated for template activity with --E. coli DNA polymerase. (w) nuclei; (-7 NAD-treated nuclei.

15

20

25

30

number

and NAD-treated Control Ca2+. template as destime and assayed untreated-control

profile of alkaline sucrose gradient of DNA obtained Figure 2. Sedimentation from nuclei incubated in a medium containing different cations and from NADtreated nuclei. Nuclei preparations were incubated for 90 min at 37O with the Control same cation combination as shown in Table I to activate the nuclei. and activated nuclei were assayed for template activity with DNA polymerase. The DNA was prepared from these nuclei and analyzed on a linear alkaline sucrose gradient described in the text. About 30 fractions of 15 drops were Top of the gradient to the right. collected from the bottom of the tube. Figure 3. Reversal of the inhibition of template-activation of nuclei treated with NAD by nicotinsmide. Nuclei were incubated with or without NAD with varied concentrations of nicotinamide for 30 min at 25'. After washing, the nuclei were subjected to the activation process as described in the text and (-> control assayed for template activity with --E. coli DNA polymerase. nuclei; (o---o) NAD-treated nuclei. 574

BIOCHEMICAL

Vol. 53, No. 2, 1973

Mg*+ as described Nuclei in which

under

preparations

fraction

were

factors

synthesis

existed.

chromatin

was determined

to DNA with ting

activation

process

min of incubation

at 37'.

activation

template

of the

can replace

Mg*+ plus

which

alkaline

were

sucrose

incubated shown

for

described

under gradient.

incubated

with (Fig.

nuclei

were

r3HlTTP

incorporation a dramatic

In the next

set of the

(Fig.

of Mg*+ plus 90 min.

DNA were

template

1).

presence

activation nuclei

90 min at 37' analyzed

be due in

Fresh

nuclei

cations

or nuclei

combination

and the through

gradient

a linear extracted

the

(Table

as

alkaline from from

[3~1~~~

fragments

as

DNA extracted

DNA obtained

Mg*+ plus

nuclei

fresh

incorporated

of DNA.

When the

Ca*+ or Mn*+ alone, I).

Parallel

of the nuclear

Ca*+,

However,

the

with

DNA in the

were

control template remained

incubated

fresh

by sedimentation

to establish

nuclei

nuclei

this

gradient

activity activity

activated

were

to

activity

40%) compared

increased

in a medium which

by NAD (1,2)

to the

at 37O in the to a maximum level

of NAD-treated

unchanged.

a relation-

The template

(about were

in an alkaline

575

induced

the nuclei.

NAD was low

template

conditions and from

of

with

the

attempted

of DNA synthesis

activity

When the

which

we have

inhibition incubated

in about

for

might profile

2).

stimulated

(Table

sedimentation

smaller

containing

Mn*+

isolated

of the

in the

of experiments

preparations

NAD-treated

that

of Mg2+ plus activity.

from

same as that

containing

90

to the medium,

template

activity

was the

In the after

2).

activation

to the

alone

added

the

DNA was prepared

same bivalent

of distribution

I).

as an activator

and its

template

(Table

of the template

(Fig,

inthat

unchanged

the addition

effective notion,

in the position

the

ted

for

was markedly shift

the observed

value

the

in a medium

between

control

Ca*+was

The DNA was centrifuged

ship

of nuclei

this

and purified

was established

in the

DNA

incorporation

the medium

However, increase

was determined

fractions

(Fig.

Mg*+ or

for

The template-activa-

remained

the activation

It was noted

finding

It

in

template

int3H]TTP system.

activity

to activation

assayed

incubated

was observed

that

Mg*+ or Ca*' with

cations

template

The pattern

2),

increase assay

in chromatin.

To test

methods.

was associated

the in the

Ca*+ and was equally

I were

sucrose nuclei

preparations

90 min at 37' with

in Table

to establish

of nuclei

was minimal.

gradient

solutions

activity

a dramatic

subjected

various

of the

When either

These results suggest I). to endonuclease activity.

was stimulated.

activation

the

caused

activity

with

bivalent

cations

RESEARCH COMMUNICATIONS

influenced

by measuring

required

Ca*+ to the medium

template

extracted

that

The template

of bivalent

nuclei

the

--E. coli DNA polymerase was found to be present

system

absence

methods,

AND BIOPHYSICAL

nuclei

subjec-

DNA was prepared from 2+ containing Mg plus Ca*+ not

activated.

sucrose

gradient,

When both their

Vol. 53, No. 2, 1973

Table

I.

Effect Activity

Cations

added*

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

of Bivalent of Isolated

Cations on the Activation Rat Liver Nuclei.

of Template

r3H]TTP incorporation+ (pmoles/lO min)

Control

5

No bivalent

cations

4

0.5 mM cap+

5

5.0 mM Mg2+

7

5.0 mM Mg2+ + 0.5 mM Ca2+

72 a2

0.5 mM Mn2+

* All of the media used contained 0.25 mM EDTA. The nuclei were activated by incubating the system at 37O for 90 min. Aliquots containing 25 ug of DNA were removed and assayed for template activity. + DNA synthesis was assayed merase for 10 min at 25'.

position

was identical

ment the

incorporated

fragments

of showed

vating

conditions

As observed with

in

the

of bacterial

with

the

with above

shift

in the

DNA poly-

above

described

fractions

finding

[ %I TTP incorporation

and a parallel

containing

the

when position

experismaller

template

of control

subjected of the

to the

acti-

DNA in the

was observed. results

an increased

clease

activity.

incubating nuclei

formation

the

template

activity

of 3' hydroxyl

primer

fragments

that this

in a medium

and Mg2+ (Table

and NAD-treated results

nuclei

suggest

To substantiate

nuclei

ribonuclease These

one unit

was associated

In agreement

an increase

The present upon

2).

?H]TTP

DNA.

nuclei gradient

(Fig.

with

II).

nuclei

suggest

that

may be a variant

the

a small

In this

preparations perhaps

of

idea,

containing

the

experiment

was activated of pancreatic

by deoxy-

the untreated-control

activated

endonuclease

deoxyribonuclease

due to endonu-

template amount

were

may be dependent

by the

present

or that

enzyme.

in the

some other

rat

liver

factors

are

operating. Purified ed nuclei coli

DNA obtained and fresh

nuclei

nuclei

activity

of DNA obtained the

activation

were

assayed

was practically

ing

that

control-activated

The template

DNA polymerase.

NAD-treated

from

from

for

activity

activated

nuclei nuclei

process.

576

template

NAD-treated-activat-

activity

of DNA obtained

identical.

DNA of untreated-control

nuclei,

On the

other

was about underwent

with

excess

from

fresh

hand,

the

20% higher, changes

during

&

and template suggestthe

BIOCHEMICAL

Vol. 53, No. 2, 1973

Table

II.

Activation NAD-treated

Type of nuclei

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of Template Activity of Untreated-Control Nuclei with Pancreatic Deoxyribonuclease.

preparation

Treatment

&ITTP incorporationg (pmoles/lOO IJ.g DNA)

None

63

Untreated-control NAD-treated

None

Untreated-control

Activation+

NAD-treated

Activation+

17

Untreated-control

DNAase*

245

NAD-treated

DNAase*

263

*Nuclei washed

and

21

were incubated without or with as described in the text.

+Template activity of the nuclei cribed in the text.

207

4 mM NAD for

preparation

40 min at 2pand

was activated

as des-

% ntreated-control and NAD-treated nuclei were incubated for 30 min at 37'in a medium containing 0.5 ug of pancreatic deoxyribonuclease, 5 mM MgCl , 50 mM Tris-HCl (pH 7.5), 0.25 mM EGTA. %he nuclei preparations were assayed for template activity with & coli DNA polymerase after the treatment as described in the text.

When ADP-ribosylation (13,14X full

medium plus plate

the

effect

extent

4 mM NAD, varied for

of control

nuclei

4 mM NAD.

proceeded

the

incorporation

rate

value.

3).

and the

At this

[14C]NAD was blocked

to the

in a and Mg 2+ The tem-

of nicotinamide

(Fig.

the inhibition

at a maximal

of

activated incubated

was assayed.

in the presence

completely control

nicotinamide

of nicotinamide

was not affected

of 30 mM prevented same as the

were

activity

incubated

with

were

nuclei

template

procedure

was the

nicotinamide

and the nuclei

concentrations

The activation

activity

was prevented

experiment

90 min and.the

to the activation

at a concentration plate

In the present

3).

containing 2+ Ca , at 37' activity

proteins

of NAD was ameliorated

(Fig.

subjected

of nuclear

and

Nicotinamide exerted by

observed

tem-

concentration

of

by about

97%.

DISCUSSION Burgoyne nuclei

for

et al.

DNA synthesis

They demonstrated endonucleolytic the

template

and that line

(15-17)

the

sucrose

that

was dependent

that activity

the

template

upon the

the activation

process

extent

was related

capacity of nicks

of isolated in the DNA.

to Ca 2+ -dependent

located

activation

in the nucleus. The present findings that 2+ 2+ Ca and Mg or Mn2+ in the medium of the DNA obtained from activated nuclei in alka-

process

sedimentation gradient

reported

showed

required

a shift

toward

577

smaller

fragments

support

the

con-

Vol. 53, No. 2, 1973

BIOCHEMICAL

tention

of the

that

activation

The endonuclease relatively (18)

responsible

tightly

reported

bound

that

had the

same cation

existed

in chromatin.

agreement

with

for

hydrolyzed

C, when Mg2'

was present

were

included

might tion

poly

reaction

in

the

enzyme was rather

Bollum

I from

the

(20) double

the medium.

mixture

activity

is

Marushige and Bonner endonucleolytic activity which

of the enzyme

(19).

template

in

showed

that

strand

copolymer,

However,

, poly

I as well

labile

the nucleus

in

pancreatic

when both as poly

as it is

poly

Mg 2+and

C were

I' Ca2+

hydrolyzed

enzyme. rat

system.

nuclease

the

was demonstrated

by isolated vating

although

report

of the

activity.

structure.

possessed

The localization

deoxyribonuclease

It

the activation

requirements

poly

by this

was due to endonucleolytic

chromosomal

chromatin

an earlier

in

template

to the

purified

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

liver

occurred

of transferase

nuclei

The block

activity. have

in the present

These

resulted appeared results

by a mechanism II

by diphtheria

ACKNOWLEDGEMENTS: This work from NICHHD. We are grateful tance.

study

that

poly(ADP-Ribose)

in an inhibition to be associated

suggest similar toxin

was supported to M. Elvira

that

of the with

template

a suppression

ADP-ribosylation

to that

formation

observed

of the with

actiof endoenzyme

ADP-ribosyla-

and NAD (21-23). in part for her

by Grant excellent

No. 5 PO1 HDO5671-02 technical assis-

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BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Marushige, K., and Bonner, J. J. Mol. Biol. 15:160 (1966). Baril, E., Brown, O., and Laszlo, J. BiochemFBiophys. Res. Conmmn. -*43.754 (1971). Bollum, F.J. J. Biol. Chem. -:2599 (1965). Collier, R.J., and Pappenheimer, A.M., Jr. J. Exp. Med. -120:1019 (1964). Honjo, T.Y., Nishizuka, Y., and Hayaishi, 0. Cold Spring Harbor Symp. Quant. Biol. 34:603 (1969). Goor, R.S., az Maxwell, E.S. Cold Spring Harbor Symp. Quant. Biol. --34.609 (1969).

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