DNA replication in isolated HeLa cell nuclei

DNA replication in isolated HeLa cell nuclei

BIOCHEMICAL Vol. 53, No. 4, 1973 DNA REPLICATION Ora McGill Received AND BIOPHYSICAL RESEARCH COMMUNICATIONS IN ISOLATED Bernard* University C...

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BIOCHEMICAL

Vol. 53, No. 4, 1973

DNA REPLICATION Ora McGill

Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

IN ISOLATED

Bernard*

University

Cancer

HELA

and

Thomas

Research

Unit,

CELL NUCLEI

P. Brent** Montreal,

Quebec,

Canada

June 22,1973

SUMMARY

DNA replication was investigated in HeLa cell nuclei isolated from different phases of the cell cycle. DNA synthesis occurred only in S-phase nuclei and was dependent on the presence of the four deoxynucleoside triphosphates, Mg*, ATP and S-phase cytoplasm. Gr-phase cytoplasm was unable to support such DNA synthesis. A purified preparation of calf thymus DNA polymerase, however, was able to replace S-phase cytoplasm in supporting ATP dependent DNA synthesis, which suggests that the S-phase cytoplasmic factor is a DNA G -phase nuclei could under no conditions be stimulated to initiate DNA ~e~KrZZFprenrraturely. In only

vitro

studies

a limited

replication. to

contribution

to

Such

replication’ We

subcellular such state. compared those

of

to for

S-phase

HeLa

for

This cell

G , -phase

report

nuclei

HeLa

in

mutants

have

regulation

made

of DNA

utilized

uiuo

celfs

integrity

of

conditionally

with

not

made

structure

defective

are

for

for

DNA

is probably

in

approach,

a

template

in

an in uivo

mammalian

cells,

cells

in order

to identify

S-phase

and

present for

analogous

replicating

G,-phase

requirements of

an

and

available

which

properties

The

enzymes

nonreplicating

the

using

nuclei.

and

replication

the

of

cells

cell

are

describes

and

have

their

mammalian

precursors

cells

and

prokaryotes

use

as template

mechanism

retaining the

in

replication

DNA

in

DNA

progress.

isolated

DNA

isolated

of the

largely with

exogenous

and

problem

yet

S-phase

necessary

Gr-phase.

this

replication

DNA-replicating

during

to

in significant

consisting

mutants

factors

from

DNA

is accessible

Since

understanding

combined

resulted

studied

system

nuclei

systems

polymerases

our

molecules’

have

have

DNA

approaches

exogenous

function.

DNA

purified

Recent

permeable and

with

during DNA

an S-phase-specific

we

replication cytoplasmic

instead

absent

in

isolated

factor

absent

cells.

METHODS

The synchronized

*Present Victoria, **Present 332 North

procedures by

for selective

Address: Australia. Address: Lauderdale,

School

culture

of

HeLa

cells

of

mitotic

derachment

of

Microbiology,

Laboratories P.O. Box

The

of Pharmacology, 318, Memphis,

Copyright 0 1973 by Academic Press, inc. At1 rights of reproduction in any form reserved.

1213

have

been

cells

described

from

University

St. Jude Tennessee

previously.’

a monolayer,3

of

Melbourne,

Children’s 38101

Cells

a method

Melbourne,

Research

Hospital,

were which

Vol. 53, No. 4,1973

BIOCHEMICAL

AND BIOPHYSICAL

30 INCUBATION

RESEARCH COMMUNICATIONS

40

TIME

(min)

Fig. 1 DNA

synthesis was assayed by incubating cell lysate equivalent to lo6 S-phase nuclei in 150 /.11 of a standard reaction mixture containing dATP, dcTP, and dGTP, each at 0.5 mM, 1.5 PM, 3H-dTTP (5 x 10’ cpm), 9.5 mM MgCls, 5 mM ATP. The reaction was terminated by addition of 5% trichloroacetic acid (TCA) at 0” C. The nuclei were sedimented by centrifugation and washed 3 times with ice cold 5% TCA. The acid-insoluble pellet was hydrolyzed by heating in 5% TCA at 100” C for 45 min. The hydrolyzed supernatant was added to Aquasol (New England Nuclear) and the radioactivity assayed by liquid scintillation spectrometry. The following additions were made after 20 min of incubation: 0, 80 ~1 S-phase cytoplasmic fraction; b, 50 PI reaction mixture; n, 20 /.d containing lo6 isolated S-phase nuclei; A, none.

does

not

involve

mitotic

index

any

metabolic

of synchronous

at 37’

C while

was

about

20

Gr , S and

Gr

or

S were

the

strokes

in

a buffer X-100;

min in mM

those

hypotonic

buffer

at

a buffer

mM

8.0;

NaCl;

120

the

four

the

S-phase

the

deoxyribonucleoside

was

1 mM

MgCla

glucose;

; 5 mM 0.05%

lysate

was

triphosphates,

were

washed

8;

by

22

centrifuging

1 mM

the

the

nuclei

pH

7.7;

a 10 in this

about

five

volume 0.1%

stage cell

to as

with

by

glucose: at this

resuspending

X-100;

open

mM

cycle

disruption

of an equal

lysate

KHsP04-KsHPO,,

triton

cell

resuspension

broken

addition

Th e cell

and

fraction,

The

After

in

cell

referred

for

by

obtained

Cells

cells

The

maintained

of the

used

being

pH

were

methods

before

restored

process.

duration

respectively.

MgCls.

Tris-HCl,

were

Cultures

2 mM

(ME).

nuclei

cytoplasmic

11 mM

cell

7 min

mM

The

replication

The

2 hr,

Mueller.4

containing

Isotonicity

DNA

S phases.

mitosis. and

7.7,

RESULTS

When

after

the 90-95%.

and

8, 10 and

at 0” C for

Isolated

containing

pH

pH

routinely

2-mercaptoethanol

cytoplasm.

(A)

were hr

disrupt

Gr

Friedman

swell

2 mM

g, removing

Tris-HCl,

by

to

120

EDTA; and

200

left

was

12

homogenizer.

containing 1 mM

2 or

buffer,

might

through phases

described

were

a Dounce

Gs

at

phosphate cells

nuclei

proceeding

harvested

essentially

mM

both

hr;

that

preparations

suspension

were

block

of triton

contained lysate

after

one

60 mM

for

1

wash

NaCl;

60

ME.

AND DISCUSSION

incubated ATP

1214

at and

37’

C with

MgCla

a reaction

, 3H-dTMP

mixture

containing

was incorporated

into

BIOCHEMICAL

Vol. 53, No. 4,1973

Table 1 Properties

of DNA

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Synthesis

Reaction

in S-Phase

pmoles Conditions

Complete

dTMP per

omit:

lo6

nuclei

12.5

dATP

0.1

dcTP

0.2

dGTP

0.1

ATP

0.1

MgClz

0.1

ATP

DNase

add :

CTP

0.2

ATP

GTP

0.2

ATP

UTP

0.2

treated

Lysate

incorporated

system

omit:

Cell

(1.5

1.8

mg/ml)

DNA synthesis during a 30-min incubation was assayed as in Fig. 1, except 3H-dTTP was 54 FM (5 x lo5 cpm). When the ribonucleoside triphosphates were added, their concentration was the same as for ATP. Deoxyribonuclease (DNase) was added at the end of a 30-min incubation period; incubation was continued for a further 30 min before the reaction was stopped with 5% trichloracetic acid.

DNA

for

up

to

deoxynucleotides, (Table

1).

that

the

We

in

background

activity of

dependence

ATP

acts

as

further,

one-tenth

the

of

the

density

much

acid-soluble

not

cell

lysate

for

ATP

system

material

was in

the

carried reaction

1215

gradient

in

occurred

of contaminating

the out

In

maintenance in the

mixture

absence was

the

same

(Table

4).

S-phase

DNA

all

analysis

Momparler

incubated

in microbial

of

four

to deoxyribonuclease

(Bernard,

was

is significant. for

presence

sensitive

isopycnic

repair

presence

the

was

synthesis

substrate reaction

and

and

the

nuclear

phosphorylation the

by

label

labeling

DNA

requirement

HeLa

required

incorporated

Gr-phase as

absolute

incorporation

replicative

When

precursors, the

and by

is

This

can be explained the

a

1).

Mg*,

synthesis

only

ATP

incubation

and

(Fig.

preparation).

view

triphosphate

ATP showed

lysate,

In

min

observed

manuscript S-phase

30

to of

of analyzed

Brent,

way

as the of

this

cells. systems,’

determine

the

DNA,

and

Most

replication

order

of

the

whether

deoxynucleoside

ATP.

After by

30

min

DEAE-ce&iose

of

BIOCHEMICAL

Vol. 53, No. 4,1973

Table

2

Cytoplasmic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Requirements Isolated

for

S-Phase

DNA

Synthesis

Nuclei

pmoles Conditions

S-Phase

cell

Isolated

S nuclei

in

dTMP per

incorporated

lo6

lysate

nuclei

4.4 + Buffer

A

1.6

+ S cytoplasm

4.2

+ G,

cytoplasm

1.7

+ G,

+ S cytoplasm

(1:l)

+ DNA

polymerase

A

+ DNA

polymerase

minus

3.6 3.8 ATP

0.2

DNA synthesis in isolated S-phase nuclei was assayed durin incubation as described in Fig. 1. Where indicated 0.5 units ofg cJf-$zk DNA polymerase A (Fraction X, Ref. 6) was added instead of cytoplasmic fraction. (A unit of DNA olymerase is the amount of enzyme catalyzing the incorporation of 1.0 nmo Pe of radioactive nucleotide into an acid-insoluble product per 10 min at 37” C.)

thin-layer Whereas the to

to

chromatography’ virtually

no

3H-thymine be

adequate

DNA

was

was

still

to

support

It appears,

reaction

and

merely

triphosphate (Table

form.

DNA

of

S-phase

synthesis

DNA

synthetic

effect

than

in

this

period

in the

of precursor

shown

synthesis

at a considerable

that

ATP

GTP,

CTP

rate

is involved

phosphorylation

3H-thymine

UTP

were

more

able

absence

of ATP,

20%

of

experiment

at least

15

min

with

the

directly

to maintain

to

as dTTP.

in a parallel

for

of deoxynucleotides

nor

remaining

replace

this

in

the

replication

them

in their

function

of

ATP

1). Isolation

for

Neither

during

of

a level

therefore,

in the

percentage

as dTTP,

DNA

of ATP.

the

synthesized

present

presence

not

determine

by

more

than

to

lOO%,

buffer

(Table

the

same

activity

of

Gt-phase

synthesis pronase

these

results in

2).

as

that

and

their 60% while

Readdition

a mixture

cytoplasm

isolated

sensitive

from

capacity

inactivity from

nuclei

S-phase

S-phase

nuclei.

ribonuclease

(Table

of

2).

Re-add

a mixture

buffer

and

due

to

the

was (Table

1216

i t’ran

of

Gr-phase of Gt-

of

contains

3),

S-phase

S-phase

cytoplasm,

presence

shown

reduced

to

suggesting

had cytoplasm

inhibitor.

heat that

capacity

labile, it

greater resulted that

We

required

restored no

indicating

an

a factor be

their

cytoplasm

cytoplasm and

S-phase

fraction

factor

insensitive

centrifugation

of

cytoplasmic This

by

re-addition of

is not

the

cytoplasm

the

conclude for

DNA

nondialyzable, is

a protein

of

BIOCHEMICAL

Vol. 53, No. 4,1973

Table 3 Properties

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of S-Phase

Cytoplasmic

Factor

pmoles

dTMP

Additions

Buffer

per

incorporated

lo6

nuclei

1.5

A

S cytoplasm

4.0

S cytoplasm

heat

S cytoplasm

treated

with

pronase

1.5

S cytoplasm

treated

with

RNase

3.5

S cytoplasm

diluted

1:l

with

buffer

A

3.0

S cytoplasm

diluted

1:2

with

buffer

A

2.4

S cytoplasm

diluted

1:3

with

buffer

A

2.0

S cytoplasm

diluted

1:4

with

buffer

A

1.6

Dialyzed

inactivated

1.6

S cytoplasm

3.6

Isolated S-phase nuclei were incubated for 30 min with the same reaction mixture as in Fig. 1. 20 ,ug pronase pretreated to inactivate DNase (80’ C for 10 min at pH 5) was incubated with 80 /.d of cytoplasmic fraction at 37” C for 10 min. 200 pg RNase was incubated with 80 ~1 of cytoplasmic fraction at 37’ C for 10 min. Where indicated, cytoplasmic fraction was inactivated by heating at 70’ C for 10 min. Dialysis was against 500 volumes of buffer A for 12 hr.

molecular with

weight

dilution

of

at saturation As

Table

the

cytoplasmic

of

our

effort

purified

in that

possibility

further,

The

that

to

(Table

3),

the

present

we

purifying

HeLa

and

the

its

activity

of

concentration

the

factor

in the

yet

replicating

DNA

for

at least

of

the

of precursors

mid

S-phase

an additional DNA

synthesis or

cofactors,

(Ref.

that

this

6)

S-phase

we

cell

tested

to replace

was

indeed

synthesis

is a DNA

HeLa

invariably

factor

polymerase’j

A

factor the

cytoplasmic

DNA

and

S-phase

nuclei

l),

cessation

nuclei, cytoplasmic

in Gr-

S-phase

calf-thymus

S-phase are

the

polymerase

S-phase

(Fig.

decomposition

Because

characterize of

incubation

premature

daltons.

fraction

calf-thymus

isolated

suggesting

polymerases

10,000

preparations

2 shows

synthesis

than

decreased is likely

not

ability

of

level. part

partially

greater

was

S-phase

able ATP

factor

to

cytoplasm.

support

dependent,

polymerase-like

cytoplasmic

the

protein. and

DNA strongly

To

analyzing

test the

this DNA

cells. stopped

cells

synthesizing

from

4 hr. in (2)

1217

which

Several the

DNA

they

explanations

subcellular

inactivation

were

after derived may

system: of

20-30

cytoplasmic

were

min

of

capable

of

for

the

be offered (1)

depletion factors,

or or

(3)

BIOCHEMICAL

Vol. 53, No. 4,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 4 Effect of S-Phase Cytoplasm

on DNA Synthesis in

Gr -Phase Nuclei

pmoles dTMP incorporated Conditions

per lo6 nuclei

Early G, -phase cell lysate

0.5

Early G, nuclei + buffer A

0.1

+ S cytoplasm

0.4

Late G, -phase lysate

0.5

Late G, nuclei + buffer A

0.2

+ S cytoplasm

0.4

DNA synthesis during a 30-min incubation was assayed as described in Fig. 1. Early Gr and late G, refers to 2 and 6 hr, respectively, after mitosis.

complete

utilization

of available template

The first of these possibilities by adding Mg*

fresh reaction

was eliminated

mixture

containing

after 20 min of incubation

after 20 min similarly fresh isolated

in the absence of initiation

failed

by our failure to prolong

the period

the four deoxynucleoside

triphosphates,

(Fig. 1). Addition

to extend

of freshly prepared

the duration

of DNA synthesis

S-phase nuclei after 20 min, however,

synthesis at the original

rate, indicating

that precursors,

at this time

if DNA

synthesis

cofactors

ATP and fraction

(Fig. 1). Addition

of

20 min of DNA

and cytoplasmic

factors do

factor resides in the nucleus.

stops because of a failure

sites.

of synthesis

cytoplasmic

did result in a further

not decay for at least 40 min, and that the limiting known

of new template

to initiate

It is not

new replication

sites or because of the decay of labile nuclear factors. All of our attempts addition

of S-phase

initiation

of replication

apparent

initiation

cytoplasm. hybrids, Kumar

stimulates population.

conflicts

Rao

and

synthesis in either early or late Gr-phase

were

unsuccessful

with

a recent report by Kumar

Johnson

throughout

(Table

4). Our

in G, -phase HeLa observed G, -phase,’

after

the rate of DNA

in contrast

observations

on the

in very early absence

1218

of

factors

of S-phase cell

system of

In is possible, therefore, cytoplasmic

S-phase nuclei

initiating

who showed

in G, /S HeLa

to the in vitro

results in terms of an S-phase-specific replication

addition

initiation

nuclei by

to demonstrate

and Friedman7

nuclei

premature

inability

where only late Gr -phase nuclei were affected.

the latter authors’

Our

DNA

synthesis

were initiated

and Friedman,

to interpret

cytoplasm

of DNA

Although nuclei

to initiate

present in

factor which in their Gr

S-phase cytoplasm,

Vol. 53, No. 4, 1973

together reside

with in the

the S-phase

ACKNOWLEDGMENTS: Calf-thymus

DNA

BIOCHEMICAL

rapid

initiation

observed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in

Gr /S

cell

hybrids,*

suggest

that

such

factors

nucleus.

This poiymerase

work

was

was

a gift

supported

by

from

R.L.

Dr.

the

National

Cancer

Institute

of Canada.

Momparler.

REFERENCES

1. 2. 3. 4. 5.

6. 7. 8.

Klein, A., and Bonhoeffer, F., Ann. Rev. Biochem., 41, 301 (1972). Bray, G., and Brent, T. P., Biochim. Biophys. Acta, 269, 184 (1972). Lesser, B., and Brent, T. P., Exp. Cell Res., 62, 420 (1970). Friedman, D. L., and Mueller, G. C., Biochim. Biophys. Acta, 161, 455 (1968). Grippo, P. Iaccarino, M., Rossi, M., and Scarano, E., Biochim. Biophys. Acta, (1965). Momparler, R. L., Rossi, M., and Labitan, A., J. Biol. Chem., 248, 285 (1973). Kumar, K. U., and Friedman, D. L., Nature New Biol., 239, 74 (1972). Rao, P. M., and Johnson, R. T., Nature, 225, 159 (1970).

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