Inhibition of ADP-ribosylation of histone by diadenosine 5′, 5‴-p1, p4-tetraphosphate

BIOCHEMICAL

Vol. 99, No. 3,198l April

AND BIOPHYSICAL

RESEARCH

15, 1981

COMMUNICATIONS Pages 837-843

INHIBITION OF ADP-RIBOSYLATION OF HISTONE BY DIADENOSINE 5',5"' -p1,p4-TETRAPHOSPHATE Yasuharu

Tanaka,

Department

Received

January

Norisada

Matsunami

and Koichiro

Yoshihara

Nara Medical University, of Biochemistry, Shijo-cho, Kashihara, Nara, Japan

lo,1981

SLJMMAR‘I

Diadenosine 5',5"' -p1,p4-tetraphosphate (Ap4A) strongly inhibited ADP-ribosylation reaction of histone by purified bovine thymus poly(ADP-ribose) polymerase.2+ This compound showed a relatively weak inhibitory effect on Mg -dependent, enzyme-bound poly(ADPribose) synthesis. Among various adenine nucleotides tested, including several diadenosine nucleotides with varying phosphate chain length, Ap4A was the most effective inhibitor of the histone-modification reaction. Ap5A and Ap6A showed slightly lower inhibitory effect than Ap4A. Kinetic analysis of the inhibitor (Ap4A) with varying concentration of substrate (NAD+) revealed that this compound is a "mixed type inhibitor", with a Ki value of 5.1 PM. Purified

bovine

vated

by binding

( 1)

and catalyzes

synthesizing

reaction

transfer

two reaction

ADP-ribosylation

attempt

to a terminal

oligo-

search

system,

we found

of this

occurring,

new inhibitors that

double

of ADP-ribose

and enzyme-bound

are preferentially to

of

can be actistranded

of

DNA

from NAD+

or poly(ADP-ribose).

conditions

of histone

polymerase

or a nick reaction

protein-bound

we reported

synthesis

thymus poly(ADP-ribose)

Recently

enzyme under which poly(ADP-ribose)

respectively(2,3).

this

diadenosine

enzyme with 5',5"'

In an these

two

-p1,p4-tetra-

This work was supported in part by a Grant in aid for Cancer Reaction (501036) and a research grant (558078) from the Minitry of Education, Science and Culture of Japan. AbbreviationszAp2A; Ap3A, AplA, Ap5A and Ap6A; diadenosine 5', 5"' -pl,p2(di-) ,-pl,p3(tri-),pl,p4(tetra-) ,-pl,p5(penta-) and -pl, p6(hexa)-phosphate, respectively. PAPP, PPAPP, PPPAPP: adenosine 5'-mono-, 5'-di-, and5'-triphosphate 3'-diphosphate, respectively. 0006-291X/81/070837-07$01.00/0 837

Copyright 0 1981 by Acadernrc Press, Inc. All rights of reproduction in any form reserved.

Vol. 99, No. 3,198l

BIOCHEMICAL

phosphate

(Ap4A),

which

polymerase

d( 4 ), is a strong

polymerase

and that

reaction

rather

than

it

is

AND

BIOPHYSICAL

known as a ligand inhibitor

specifically the

RESEARCH

COMMUNICATIONS

of a subunit

of DNA

of poly(ADP-ribose) inhibits

auto-ADP-ribosylation

histone of

modification

this

enzyme.

MATERIALS AND METHODS 3H]NAD+ was purchased from New England Materials [Adenine-2,8Diadenosine compounds (Ap2A, Ap3A, APIA, Ap5A and Ap6A), Nuclear. Adenosine, S'ADP, ADP-ribose and NAD+ were obtained from Sigma. 5'-AMP, and 5'-ATP were obtained from Kojin Co., Ltd., Tokyo. Other adenine nucleotides (pApp, ppApp and pppApp) were products of Sanraku-Ocean Co. Ltd., Tokyo. Preparation for "active DNA" "Active DNA", aDNA copurified with bovine thymus poly(ADPxbose) polymerase (5 ) was prepared acceding to the method described previously (6 ). Although we presumed, in early studies (5 ,6 ), that the high enzyme-activating ability may be due to its specific base sequence, recent studies for the enzyme-activation by DNA (1 ,7 ) has suggested that DNA breakages (nicks) concentrated on the DNA may The presence of simibe the main reason for its high activity. small DNA fraction in poly(ADP-ribose) polymerase preparation lar has been reported also by Nidergang et al. ( 8 ). Enzyme Preparation Bovine Thymus poly(ADP-ribose) polsmerase with a purity of appoximately 97% was prepared according to the method described in a previous report ( 5). Enzyme Assay The Mg2+-dependent and histone-dependent enzyme reactionwas performed as described in a previous report ( 2 ). The Mg2+-dependent reaction mixture contained 25 mM Tris-HCl buffer, pH 8.0, 0.5 mM dithiothreitol, 10 mM MgC12, 0.2 pg of active DNA, 20 PM [3H]NAD+ (20 cpm/pmol) and 0.1 pg of purified poly(ADP-ribose) polymerase in a total volume of 0.2 ml. The histone-dependent reaction mixture contained 20 pg of purified histone Hl in place of Mg2+ in the reaction mixture described above. The reaction was carried out for 10 min and 2.5 min for Mg2+- and histone-dependent reaction, respectively. Other procedures were described in a previous report ( 2). In the histone-dependent reaction, at least 70% of the product was oligo(ADP-ribose) bound to histone (2 ), while almost all reaction product synthesized under the Mg2+-dependent reaction condition was enzyme-bound poly(ADP-ribose) ( 3). PEI-cellulose Thin Layer Chromatography. PEI-cellulose TLC of Ap4A (0.25 ~01) was perfomed accordinq to the method of Randerath et al . ( g)'with a solvent of 1 M LiCl; After desalting with methanol, the plate was dried and cut into pieces with 1.0 cm width. Each pieces were extracted with 5 ml of 3 N NH40H. After lyophilization of the extract, each sample was dissolved in 2.6 ml of distilled water. RESULTS AND DISCUSSION With systems

the

use

we tested

of histone-dependent the effect

of Ap4A.

838

and Mg2+ -dependent As shown in Fig.

reaction l.,

Ap4A

BIOCHEMICAL

Vol. 99, No. 3,198l

01

0

II.2

0.1

0.3

AND BIOPHYSICAL

0.4

Fig.

Effect

1.

of

0

02

iwiA c,"M) As

0"

IO

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20

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50

I00

ADEEIII'E NUCLEOTIDES (y'l)

and Q 2+ -dependent

Histone-dependent

Reaction,

The enzyme reaction was performed as described under Materiala and Methods except that the reaction mixture contained the indicated concentration of Ap4A. The respective enzyme activity g/10 min and 199 pmol/O.l pg/2.5 min without Ap A (213 pmol/O.l for the Mg $+ -dependent and t' istone-dependent activity, respectively) was set at 100%. open and colsed circles: Mg2+- and histone-dependent enzyme activity, respectively. Fig.

2.

Enzyme thods with ration of reaction Ap5A, LA;

Effect of Various Diadenosine Nucleotides on --~ the HistoneDependent Enzyme Reaction. assay was performed as described under Materials and Mehistone-dependent reaction mixture. Varying concentdiadenosine nucleotides as indicated was added into the mixture. O--O; Ap2A, a---O; Ap3A, L+--0; APIA, A--A: Ap6A.

strongly

inhibited

compound

was

modification

much

for

dependent

half

length

of

was

we

tested

diadenosine (Table

the

1).

to enzyme.

The concentration

10 )IM and

to see whether other

related

nucleotides Although

in the histone-dependent

dependent

reaction,

diadenosine

were the most potent

adenine

with all

this

of

reaction nucleotides

inhibitors

839

autoof Ap4A

than

inhibition

400

is

nucleotides

different adenine

more

this

and Mg2+ -

of the histone-dependent

effective

residues

reaction, while 2+ Mg -dependent,

the

approximately

In order

Ap4A,

several

inhibitory

inhibition

reaction

respectively. to

less

reaction

required

fic

the histone-dependent

TM,

speci-

including

phosphate nucleotides

chain were

more

the Mg2+ -

than

in

with

4 to

6 phosphate

of the histone-dependent

BIOCHEMICAL

Vol. 99, No. 3,1981

Table 1.

Effect

AND

BIOPHYSICAL

RESEARCH

of Adenine Nucleotides

on Enzyme Activity

Enzyme Activity nucleotides (0.1 mM)

histone-dependent reaction

none

Mg2+-dependent reaction 100

adenosine 5'-AMP 5'-ADP 5'-ATP

90 84 64 55

100 91 93 92

Ap2A Ap3A Ap4A Ap5A Ap6A

50 34 12 14 11

92 80 72 84 73

PAPP PPAPP PPPAPP

78 69 63

103 106 100

Enzyme assay was performed as described

into

The indicated

the reaction

nucleotide

reaction, for

the

of nucleotides

The enzyme activity

compounds tested. of

pound was carried

out.

nucleotides

pounds with

under Materials

and

was added

obtained

with no

was set at 100%.

among the

adenosine

concentration

mixture.

inhibition

longer

effective

inhibitor

suggest

a specific

Ap4A.

Fig.

Although (Fig.

phosphate

chain

than

affinity

and histone

of the

DNA and 20-fold

reaction

decreace

by these

curves

a tendency

com-

of various

that

are more effective

the

di

com-

inhibitors,

Ap5A and ApGA, and was the most tested

compounds.

of poly(ADP-ribose)

3 shows the effect

examination

reaction

dose responce

2) revealed

among the

(enzyme activator)

Thus further

the histone-dependent

Ap4A was more inhibitory

inhibition

(%)

100

Methods.

COMMUNICATIONS

of Hl

by Ap4A. in histone

840

increasing (acceptor

These results polymerase concentration

of ADP-ribose)

Though a 25-fold Hl concentration

for of DNA on the

increase apparently

in

BIOCHEMICAL

Vol. 99, No. 3,198l

AND

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RESEARCH

IO

04 Fig.

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20

30

40

50

I/S (ntl)

3.

Effect of Increasing Concentration Active DNA and Histone ~-Hi-6ii-t-s Inhibition by Ap4A. --A: The inhibition by Ap4A was examined in the reaction mixture containing a fixed concentration of histone Hl (20 pg/O.2 ml) and varying concentration of active DNA (O-o; 0.2 ug, 0-O; 0.1 pg, and +A; 5.0 pg). B: The inhibition by Ap4A was examined in the reaction mixture containing a fixed concentration of active DNA(0.2 ug) and varying concentration of histone Hl (AA; 0.1 pg, 0-O;

5 pst o---o;

20 pg).

In both experiments (A and B), enzyme assay was performed under the histone-dependent reaction condition, as described under Materials and Methods. The indicated concentration of Ap4A was added into the reaction mixture. The respective enzyme activity obtained without inhibitor was set at 100%. Fig.

4.

Double Reciprocal Plot ~/s(NAD+) in ---___ of l/v versus -- the Presence of Inhibitor. Enzyme assay was performed with varying concentration of NAD+ in the absence (O-O) or presence of 10 JJM(o---O) and 30 PM (A-A) of Ap4A in the histone-dependent reaction mixture as described under Materials and Methods. Double reciprocal plots of velocity (nmo1/2.5 min/O.l pg) versus substrate concentration (NAD+, mM) are shown.

did not of

affect

these

changes

observation dent

the

slightly

preferentially

DNA ratio

In order

to obtain

reciprocal

ration

was examined (Fig.

4).

of Ap4A so remarkably, the

inhibition

to our previous occurs

one that

at a relatively

(Fig.3).

both This

histone-depenhigh

histone

( 2 ).

double

PM Ap4A

activity decreased

may be related

reaction

Hi/active

inhibitory

the kinetic

plot

parameter

of velocity

versus

of the

inhibition,

substrate

(NAD+) concent-

in the absence or the presence

of

As shown in this

plots

841

figure,

three

10 and 30 crossed

BIOCHEMICAL

Vol. 99, No. 3,198l

AND

BIOPHYSICAL

DISTRNCEFROMORIGIN

RESEARCH

COMMUNICATIONS

(CM)

Fig.

5. PEI-cellulose Thin Layer Chromatography of Ap4A. Ap4A (0.25 ~01) was separated by chromatography on a PEIcellulose thin layer plate as described under Materials and Methods. After the samples were extracted from PEI-cellulose plate and prepared as described under Materials and Methods, the absorbance at 260 nm (white columns) and the inhibitory activity on the histone-dependent reaction (black columns) were examined. In order to assay the inhibitory actibity, 50 ~1 of each sample was added into the histone dependent reaction mixture and enzyme activity (0.1 ug) was assayed: the activity without inhibitor was set at 100% and inhibition was expressed by % decrease of the activity. A schematic illustration of the spot of markers (5'AMP, 5'-ADP, 5'ADP and Ap4A on the TLC plate is shown at the bottom of the figure.

at a point

and showed different

indicating

that

Based on these

this

compound (Ap4A)

values

Ap4A was calculated In order

parative graphy

in the

was carried plate

that

When the

was recovered

on TLC plate

at

After

with

activity

absorbance

and Vmax,

type

plots,

inhibition obtained

(PEI)-cellulose

was cut

of each fraction

activity

the

out.

activity

the

a mixed

from these

commercially

was extracted

and Methods.

is

Km values

inhibitor.

the Ki value

of

to be 5.1 PM.

polyethyleneimine

PEI-cellulose pieces

obtained

to confirm

a contaminant

apparent

of Ap4A is not

due to

Ap4A preparation,

pre-

thin

development

into

pieces

with

layer

of

the

position

of Ap4A was also

the

figure

confirmed

842

themain of Ap4A,

260 nm and a schematic

at the bottomof

under

the

and each Materials

at 260 nm and inhibitory

was examined, at the

sample,

1 cm width

3 N NH40H as described absorbance

chromato-

peak of inhibitory which

illustration (Fig.

5).

is

shown by

of

the

spot

The inhibitory

by a chromatographic

separation

vol. 99, No. 3,198l

(DEAE-cellulose to the not

procedure

BIOCHEMICAL

AND BIOPHYSICAL

column chromatography) described

by Rapaport

of

RESEARCH

this

COMMUNICATIONS

compound according

and Zamecnik

(10 ) (data

shown).

REFERENCES H., Yoshihara, K., and Kamiya, T.(l980) 5. Biol. Chem., 1. Ohgushi, 255, 6205-6211. H., and Yoshihara, K. (1979) 2. Tanaka, Y., Hashida, T., Yoshihara, J. Biol. Chem., 254, 12433-12438. 3. Yoshihara, K., Hashida, T., Tanaka, Y., Matsunami, N., Yamaguchi, A and Kamiya, T. (1981) 5. Biol. -Chem., in press. 4. G&unt, F., Waltl, G., Jantzen, H.M., Hamprecht, K., Huebscher, U and Kuenzle, C.C. (1979) Proc. Natl. Acad. Sci. USA, 76, ----6&-6085. 5. Yoshihara, K., Hashida, T., Tanaka, Y., Ohgushi, H., Yoshihara, H and Kamiya, T. (1978) J.dBiol. =., 253, 6459-6466. 6. Haihida, T., Ohgushi, H., Yoshrhara, K. (1979) Biochem. Biophys. Res. Commun., 88, 305-311. Tanaka, Y., Yoshihara, H., Hashida, Y., Ohgushi, 7. Yoshihara,., H., Arai, R., and Kamiya, T. (1980) Novel ADP-ribosylation of Regulatory Enzymes and Proteins, Developments -in Cell Biology_, Vol. G.(Smulson, M.E., and Sugimura, T., eds), ~~33-44, Elsevier/ North-Holland, New York. C., Okazaki, H., and Mandel, P. (1979) Eur. 5. 8. Niedergang, Biochem., 102, 43-57. K., Janeway, C.M., and Stephenson, M.L. (1966) 9. Randerath, Biochem. Biophys. Res. Commun., 24, 98-105. E., and Zameknik,.C. (1976) Proc. Natl. Acad. Sci. 10. Rapaport, USA, 73, 3984-3988.

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