DNA polymerases and DNA topoisomerases solubilized from nuclear matrices of regenerating rat livers

Vol. 124, No. 3, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

November 14, 1984

Pages

DNA POLYMERASES AND DNA TOPOISOMERASES SOLUBILIZED REGENERATING RAT LIVERS Miwako Laboratory

Nishizawa,

Kazushi

Tanabe*

917-924

FROM NUCLEAR MATRICES OF

and Taijo

Takahashi

of Biochemistry, Aichi Cancer Center Research Chikusa-ku, Nagoya 464, JAPAN

Institute

Received October 1, 1984 SUMMARY. DNA topoisomerase activity together with the activities of DNA polymerase were detected in a form tightly associated with rat liver nuclear matrices. DNA polymerase activities were solubilized from the nuclear matrices of regenerating rat livers by sonic treatment followed by extraction of these activities with detergent and salt. The predominant activity was mainly apolymerase as judged from the size determined by sucrose density gradient centrifugation. However, only 8-polymerase activity was detected in the matrix of normal rat livers. DNA topoisomerase activity, detected in both regenerating and normal liver nuclear matrices, showed a molecular size of about 4 S in sucrose gradient, and was active in the presence of EDTA. These results suggest that this enzyme belongs to type I topois0merase.e 1984 Academic press. I,,~.

The nuclear NaCl,

matrix,

was reported

sequence

Smith

bound

to the

nuclear on the

in this

(15,161.

a set

anchored

of enzymes

replication factors

is

should

may be organized

DNA synthesizing all

in the

or at least

nuclear other also

of these

the end of a DNA replicating

than

the

the

(3,5).

(IO-

rat

liver.

cells

that

by

of the enzyme DNA replication

According

to this

hypothesis,

involved

in the

DNA

in the matrix. or "replisomes"

as shown by Smith

enzymes

and origin

a was tightly

existence idea

2M

sequence

regenerating

a-polymerase

complexes

phase of the cell a part

matrix

gene

of salt-treated

showed

support

be contained

in large

from

with

DNA (l-6),

DNA polymerase

staining

studies

nuclei

transcribed

that

also

isolated

synthesized

prepared

antibodies These

and factors

process

they

immunofluorescent

monoclonal

machinery

reported

that

anti-a-polymerase structure

newly

the

and actively

(13,141

matrix

treating

with

(7-91,

and Berezney

reports

after

to be enriched

of DNA replication

12).

Recent

obtained

may be released

These (3)

and Berezney from

enzymes

prior

and

to the

(171,

the matrix

but after

phase.

* To whom correspondence should be addressed. EDTA, ethylenediaminetetraacetic Abbreviations: sulfonyl fluoride.

acid;

PMSF, phenylmethane-

0006-291X/84 $1.50 917

Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form resewed.

BIOCHEMICAL

Vol. 124, No. 3, 1984 In this a-polymerase

report,

we showed

tightly

bound

artificial

solubilization

sonication

followed

the

to the of both

by detergent

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

presence nuclear

of DNA topoisomerase matrix

enzyme activities and salt

of rat from

liver,

together

with

and the

the matrix

by using

treatment.

METHODS Preparation of nuclei: Wistar rats (270-300 g, Charles River) were partially hepatectomized 24h prior to liver isolation. Minced tissue was homogenized by a Potter-Elvehjem homogenizer with four volumes of buffer A (5 mM Tris-HCl, pH 7.6, 0.25 M sucrose, 5 mM MgC12). After filtration through four layers of cheesecloth, the homogenate was centrifuged at 1,800 rpm (450 xg) for 10 min, then washed twice with buffer A containing 0.5% Triton X-100 followed by washing twice with buffer A without detergent. The crude nuclear pellet was resuspended in about 10 volumes of 2.0 M sucrose containing 5 mM After centrifugation at 25,000 rpm (82,000 xg) for Tris-HCl and 5 mM MgC12. 60 min using a Beckman SW 28 rotor, the nuclear pellet was washed twice with buffer A. Preparation of method described by nuclei (37°C for 30 pH 7.6, 0.2 mM MgC12, (LS buffer plus 2 M 100, and twice with 10 min.

nuclear matrices: Nuclear matrices were prepared by the Smith and Berezney (14). In brief, endogeneously digested min) were extracted 4 times with LS buffer (10 mM Tris-HCl, 1 mM PMSF), followed by 3 times extraction with HS buffer NaCl), then once with LS buffer containing 0.5% Triton XLS buffer. Centrifugations were 5,000 rpm (3,000 xg) for

Reaction mixture of 25 ~1 contained the following: DNA polymerase assay: 50 mMTris-HCl H 7 4 at 37"C, 6 mM MgC12, 1 mM dithiothreitol 100 $l each of dATP, dGTP indp d&P, 50 l&l [3H]dTTP (120 cpm/pmol), 12% (v/vj glycerol , 400 W/ml bovine serum albumin, 160 ug/ml activated calf thymus DNA, and 2 or 4 ~1 of enzyme fraction or suspension of nuclear matrices. After incubation for 30 min at 37"C, the reaction mixture was transferred to a DEAE-cellulose paper disc (Whatman DE81), and washed as described by Lindell --et al. (18). DNA topoisomerase assay: Assay no. 1 for type I topoisomerase: The reaction mixture (10 1) contained the following; 25 n+l Tris-HCl, pH 8.0 at 3O"C, 40 mM KCl, 1 mMUEDTA, 0.5 mM dithiothreitol, 30 ug/ml bovine serum albumin, 0.5 ug colicin El DNA (Nippon Gene Co., Toyama, Japan), and 1 or 2 ~1 of enzyme fraction or nuclear matrix suspension. Assay no. 2 for type II topoisomerase: The reaction mixture (IO ~1) contained the following; 25 mM Tris-HCl, pH 8.0 at 3O"C, 40 mM KCl, 10 mM MgC12, 0.5 mM dithiothreitol, 3Opg/ ml bovine serum albumin, 1 r@i ATP, 0.5 pg colicin El DNA, and 1 or 2 J of enzyme fraction or nuclear matrix suspension. After incubation at 30°C for 15 min, the reaction was stopped by the addition of 2.5 ~1 of 5% sodiwndodecyl sulfate, 50% glycerol, 0.1% bromphenol blue. The reaction mixture (10 ~1) was loaded to a 0.8% agarose gel (10x6x0.3 cm), and electrophoresed for 2.5h at 50 volts constant in 40 mM Tris-acetate, pH 8.4, 1 mM EDTA at room temperature, The gel was stained in 0.5 pg/ml ethidium bromide, and the DNA bands were visualized by illumination with a ultraviolet lamp. The gel was photographed with Polaroid type 665 film. Solubilization of enzymes from the nuclear matrices: Nuclear matrices suspended in LS buffer were kept at 0°C for 24h and disrupted by sonication using Kontes sonicator at maximum setting for 5 times at 20 set intervals. After the sonication, 20% Triton X-100 and 4 M KC1 were added to the suspension to make final concentrations of 0.5% and 0.5 M, respectively. After mixing, 918

Vol. 124, No. 3, 1984

BIOCHEMICAL

the suspension was kept (10,000 xg) for 10 min.

at 0°C for

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5 min,

then

centrifuged

at 10,000

rpm

Sucrose density gradient centrifugation: A sample of 0.2 ml of the above prepa) % sucrose density gradient containing 50 mM Tris-HCl, pH 7:7,m0.! mM EOTA, 1 mM dithiothreitol, and 0.5 M KCl. Centrifugation was carried out at 45,000 rpm (190,000 xg) for 15h by using a Beckman SW-55 rotor, after which fractions of 0.22 ml were collected from the bottom of the centrifuge tube. Catalase (11.2 S), aldolase (7.6 S), bovine serum albumin (4.4 S), and ovalbumin (3.6 S) were used as internal markers for the determination of the sedimentation coefficient. Other diphefiine determined

methods: Amounts of DNA and RNA were determined by standard and orcinol reactions, respectively. Protein content was by the dye binding assay using Bio-Rad Protein Assay.

RESULTS Characterization

of isolated

From our preliminary the nuclei

the

these

(data

studies

taken

nuclei those

of normal

of isolated

nuclear

protein,

nuclear

RNA.

the

reached

its

level

preparations

previously

contents

of the

of a-polymerase

maximum 24h after

to use the

the

of nuclear

liver

matrices

high

in

partial

24h regenerating

described

These

nuclear

and regenerating

nuclei.

about

used in

salt-treated

final

The matrix

1% of the

values

are

rat

total

matrices liver

were

contained

nuclear

in agreement

prepared analyzed

about

from and compared

10% of the

total

DNA, and 20 to 30% of the total with

the data

of other

investiga-

(19-22).

Characterization

of DNA polymerase

The matrix its

activity

(see METHODS).

purified

tors

All from

DNA, RNA and protein

with

liver

the

We decided

below.

were

preparation

rat

not shown).

experiments

matrices

experiments,

of regenerating

hepatectomy for

nuclear

bound

template-primer

This

activity

Fig.

1 shows that

results

DNA polymerase

was about

was characterized

compared

suggest

by using

to aphidicolin

that

with the

the

to the

activity

10% of the

the DNA polymerase

was more sensitive dideoxy-TTP

bound

total well

activity

nuclear

measured nuclear

predominant

exogeneous

known specific

DNA as

activity.

inhibitors.

in regenerating

liver

matrices

and more resistant

in normal

DNA polymerase 919

with

polymerase

and N-ethylmaleimide, enzyme activity

matrices

bound

liver

matrices.

to the

to These

regenerating

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 124, No. 3, 1984

regmerating

Fig.

liver

1.

liver matrix

Inhibitor characterization of DNA polymerase activities of nuclear matrices of normal and regenerating rat livers. The indicated amount of inhibitor was added to the reaction mixture, and the remaining DNA polymerase activity was measured. A: aphidicolin; the concentration of dCTP and dTTP was 20 fl each. B: dideoxy TTP; the concentration of dTTP was 10 uM, C: N-ethylmaleimide; dithiothreitol was omitted. Other reaction conditions were the same as the standard assay described in METHODS. 0: normal liver matrix, e regenerating liver matrix

matrices

is a-polymerase

whereas

of DNA topoisomerase

activity

that

of the

normal

liver

matrices

is

$-polymerase. Detection

DNA-relaxing normal

activity

tightly

and regenerating

rat

El DNA as a substrate. conditions reaction

for

however,

with

method

of a large

the

is

unable

excess

Solubilization

extracted

activity

to detect

is type of the type

matrices

by using

supercoiled

detected

molecular

I topoisomerase.

presence

of type

II topoisomerase

colicin

in both

As judged

predominant

in both

reaction

from

the

species

We cannot,

II enzyme, activity

since

in the

from

nuclear

and DNA topoisomerase matrices

tightly

matrices

activities bound

920

to the

remaining structure

our

presence

I enzyme.

of enzyme activities

nuclear

the

matrix

nuclear

was equally

that

matrix

possibility

of type

DNA polymerases salt

was detected

we concluded

the nuclear

exclude

to the

I and type11 DNA topoisomerases.

properties,

associated

assay

type

bound

livers

This

in the nuclear

in the high and was diffi-

Vol. 124, No. 3, 1984

A IL-

BIOCHEMICAL

5

Fig.

4. 3.6s

10

15

fraction

cult

I

26

1

2.

by sonication, of the

of these

of the

solubilized

pattern

of the

These to the shown showed

results

clearly

in panel only

number

a main

B, the

matrices

polymerase

one peak at about

could

sucrose

from

that

2.

gradient

Panel

the

activity

predominant

extracted f%polymerase.

921

applied. by using

a

centrifugation

A shows the rat

liver.

at 7.5 S and a minor

is a-polymerase.

3 S; i.e.

separately

be achieved

density

regenerating

peak

when

in METHODS.

shown in Fig.

demonstrate liver

however,

of the

enzymes

two peaks;

or salt

as described

are

solubilized

regenerating

enzymes,

patterns enzymes

showed

detergent,

treatments

The sedimentation

activities

fraction

Sucrose density gradient analysis of DNA polymerase and DNA topoisomerase solubilized from nuclear matrix. Panel A: extract from regenerating liver matrix. Panel B: extract from normal liver matrix. top: DNA polymerase activity; bottom: DNA topoisomerase activity measured by assay no. 2. I and II indicate the position of form I (supercoiled) and form II (relaxed) colicin El DNA, respectively.

solubilization

combination

20

number

to solubilize

Partial

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

activity

ONA polymerase peak at 3 S.

DNA polymerase On the other from

normal

hand, liver

bound as matrices

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 124, No. 3, 1984 The main activity of about

4 S, which

Topoisomerase ating

liver

bottom

peak of DNA topoisomerase was the

activity

of this

matrices,

but

liver

matrix.

expected size

a minor

of the centrifuge

ating

size

tube

activity

for

type

was detected

DNA relaxing

only

size

in both activity

of this

was not detected

at a position

I topoisomerase

(22-24).

normal

and regener-

was detected

in the solubilized

The molecular

and DNA polymerase

was sedimented

sample

activity

in this

at the

of the

regener-

was more than

11 S,

fraction.

DISCUSSION Several the nuclear

lines matrix

Pardoll

--et al.

nuclear

matrix.

process

involving

theorized

Since

that

sites than

the presence (13-17,25).

is

of enzymes

on the nuclear a-polymerase

sites

--et al.

were

matrix

must

these

in DNA replication,

and to the

multi-step

factors,we contain

a in

(5)

attached

known to be a complex

and protein

involved

of DNA polymerase

Berezney

DNA replication

DNA replication

many kinds

other

concerning

have been reported

(3)

DNA replication enzymes,

of evidence

believe

that

enzymes,

but

were

found

no

in

the matrix.

In this

study,

together

with

from

liver.

rat

we have presented

DNA polymerases

Topoisomerases strand

break

a double

about

break

in the

this

100 kDa by proteolysis to be a dimer

we employed

sonication,

the

the combined

was 50 to 60% of that

of the

starting

matrix

prepared

II topoisomerase Eukaryotic

makes

topoisomerase

and has been reported

may be derived on the

other

to be

from the

enzyme of

hand,

was reported

(27). enzyme activities

treatments

extraction.

activities

I enzyme makes a single

and type

sources

Type II enzyme,

to solubilize

and KCl-Triton

several

nuclear

type

of ATP (26).

enzyme species

of 172 kDa polypeptides

It was hard until

presence

ATP,

DNA topoisomerase

to the

in two types;

from

(23).

that

bound

DNA without

been purified

67 kDa, whereas

tightly

classified

on supercoiled

strand

has previously

are

are

evidence

from

of hypotonic Recovery

matrix.

922

the

nuclear

incubation,

of DNA polymerase

matrix extensive activity

I

BIOCHEMICAL

Vol. 124, No. 3, 1984 The matrix order

extract

to separate

The predominant polymerase,

merase level

was subjected

the molecular

as expected,

of a-polymerase

regenerating

and the main

detected

density

rat

topoisomerase

expected

in the normal was detected.

gradient

of DNA polymerase

in the

4 S, the position

was also

to sucrose

species

DNA polymerase

to be at about

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

for

liver These

liver

type

I enzyme.

matrix,

although are

of Duguet

--et al.

(28);

they

demonstrated

that

topoisomerase

activity

in rat

liver

nuclei

was not

significantly

the

process

In addition activity

regenerating

a molecular increase

hepatectomy,

and suggested

We believe

that

topoisomerase

induced

possibility

that

The role not clear, the

clues

this

this

than

this

by partial

in understanding

11 S.

showed

its

peak

Type I topoisoonly

the

a very with

level

low

the of type

I

changed

hepatectomy,

of the the

in

et al.

but we cannot

of these

gradient

reported

after

a

partial

in DNA replication.

in the nuclear

enzyme in the

(28)

nuclei

involved

form

in the eukaryotic

DNA relaxing

of the sucrose

liver

or a complex

function

another

bottom

Duguet

enzyme is

an aggregate

the existence

at the

enzyme activity

of DNA topoisomerases

but

detected

II topoisomerase

that

sizable

matrix

larger

of type

is

we also

liver

size

considerable

was a-

regeneration.

to the 4 S activity,

in the

indicating

of liver

matrix

consistent

observations

during

in

and topoisomerase.

activity

results

analysis

matrix

is type

exclude

of type

matrix

the

I enzyme.

DNA replication nuclear

II

is still may be one of

enzymes.

ACKNOWLEDGEMENTS This research was supported in part by a Grant-in-Aid for from the Ministry of Education, Science and Culture of Japan. I. Inagaki for preparation of the manuscript.

Cancer Research We thank Miss

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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