Calcium-dependent priming of DNA synthesis in isolated rat liver nuclei

Vol.39,No.2,

1970

BlOCHEMlCAlANDBlOPHYSlCALRESEARCH

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CALCIUM-DEPENDENT PRIYING OF DNA SYNTHESIS

IN ISOLATED RAT LIVER iilUCLEI* Leigh

A. Burgoyne,

School

M. Anwar Waqar and Maurice

R. Atkinson

of Biological Sciences, Flinders University of South Australia, Bedford Park, South Australia 5042.

Received March 2, 1970 SUMMARY : Nuclei isolated from rat liver in the absence of Ca2+ incorporate deoxyribonucleotides into added poly d(A-T) but not into their nuclear DNA, which is of high molecular weight. On exposure to Ca2+ the nuclear DNA is nicked and then serves as primer for incorporation of deoxyribonucleotides, in the presence of Mg2+, through the action of nuclear DNA polymerase. Incorporation of the chain-terminating arabinosyl analog araCMPI is a measure of Ca2+-dependent production of DNA primer sites in nuclei. Although (cf.

1,2)

little which

known of the

nuclear nuclei

that

from

Escherichia

3'-hydroxyl

primer

DiJA replication primer that

is

ends

are

is

unable

ed in a Ca2+-dependent

of studies

control

controlled

now being

prepared

from

were

homogenized

adult

to initiate

A - 1.37

nick

their

that

the Dp+A and

describes

until

at nicks,

The possibility that

report

DNA synthesis

(3-8), and repair.

I)NA synthesis

IXA (9).

This

nuclei

isolation

of

DNA has been nick-

reaction.

chloride,

pH 7.4)

rat

(10)

liver

rapidly

EDTA, 0.5 mM EGTA/g Buffer

isolated

by endonucleases studied.

DNA polymerases

DijA replication

initiates

ends in duplex

- Isolation, washing --of Nuclei (GO mM KCl, 15 mM NaCl, 0.15 mM spermine, 15 mM tris

in

nuclear coli

Isolation etnanol,

of nuclear

incorporation

factors

D1\IA polymerase provide

generate

have been a number

and of deoxyribonucleotide

is

Purified

there

in at least

of liver

M sucrose,

and assay were 0.5

with

with

the

carried

mM spermidine,

additions follo\!ing

7 ml of Buffer

and the homogenate

out in Buffer 15 mM 2-mercapto-

as indicated.

Nuclei

modifications. A - 0.34

was layered

Livers

M sucrose, on 0.33

1 mM EDTA, 0.25 mM EGTA and centrifuged

were

2 mM

volumes for

sucrose,

of

15 min

at 16,000 g. buffer

P

The nuclear pellet was dispersed in 7 volumes of Buffer A - 2.4 M 0.1 mM EDTA, 0.1 mM EGTA, layered over an equal volume of the same

and centrifuged

for

45 min at 75,000

* This work Committee

g.

After

one wash in Buffer

A -

was supported by grants from the Australian Research Grants and the University of Adelaide Anti-Cancer Foundation. analogs of CMP and CTP; EGTA : ethylene ' araCMP, araCTP : arabinofuranosyl glycol-bis-(2-aminoethyl ether)-N,N'-tetraacetic acid. 254

Vol.

No.2,1970

BIOCHEMICAL

40

20

RO

40

60

AND BIOPHYSICALRESEARCH

-

50

-

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Minutes

50

Minutes

Minutes

Figure 1. All assays were in 50 ul of Buffer A - 0.34 enol pyruvate, 0.4 mM ATP with nucleotides as indicated. and incorporation of 32 P into DNA was measured by scintillation addition of 10 ~1 of 10 mM EDTA, 1.8 M KOH and removal of by washing on paper discs with 10% CC1 COOH; 5 times with CC13COOH, 1.5 mM KH2P04; and twice WI4 h ethanol.

100

100

\1 sucrose, 2 mM phosphoIncubations were at 37O counting after acid-soluble material 0.25 Y Na2S04, 5%

A. Nuclei (5.0 x 106/assay) in assay solution with 1 r\l EDTA 0 2 mM E TA, 10 mM MgC12, 0.4 mM each of dATP, dCTP, dGTP, and 16 UM a- 3yP-dTTP'(4:0 x log cpm/umole): t, no additions; 1 mM CaC12 at zero time.

Q,

+ poly

d(A-T),

90 url in residues;

+-,

4 mM each of dl\.TP, dGTP, and dTTP; Q, + .-32P-dTTP + 0.4 mM Activated nuclei (2:8 x 105/assay in assay solution with 1 mM EDTA, EGTA, 10 mM MgCl2: +, + 27 UM a- 3*P-dTTP (2.7 x 109 cpm/vmole); 0, P-dTTP and 0.4 mM each of dATP, dCTP and dGTP. Activated nuclei (8 x 105/assay) in assay solution with 1 mM EDTA, 0. mM EGTA, 10 mM MgC12, 0.4 mM each of dA P, dGTP and dTTP: a, + 3.2 PM a- i' *P-araCTP (1.2 x 109cpm/umole); 0, + '5*P-araCTP and 1 mM CaC12.

255

+

Vol.39,No.2,

3.34

M sucrose

persed where

1970

for

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

by centrifuging

assay

indicated, for

were

washed

--Results tained

an active

dGTP,

.-32 P-dTTP

In these

g, the nuclei

(2 ml/liver).

Nuclei

were

were

dis-

activated,

in Cuffer A - 0.34 :I sucrose, 1 mM CaC12, 5 mF1 Ca2+ and Fig2+ were then removed by centrifuging of suffer A - 1.0 V sucrose, 2 mtl EDTA, 0.5 tnfl EGTA. The

10 min.

and w-suspended

and Discussion

Nearest-neighbor shown),

in the same solution

an underlay

nuclei

15 min at 16,030

by suspending

MgC12 at 0-2O through

for

conditions but with

- Nuclei

in Buffer isolated

in

F. - 0.34 (Ca2+,

:I sucrose.

11g2+) - free

solutions

con-

UijA polymerase when assayed with poly d(D-T), df,TP, dCTP, 2+ , with EGTA to selectively chelate Ca 2t (Fig. IA). analysis showed all label in alternating d(A-T) copolymer.

and Mg

added no added

calf primer

thymus

Dl!A also

CNA synthesis

served

as primer

was at tne limit

(results

not

of detection

I -

Figure 2. Nuclei (3 x 106/assay) in 100 ~1 of assay solution as for F' l9 with 1 mM CaCl 9 mM MgCl 0.4 mM each of dATP, dCTP, dGTP and 18 UM U- 39 P-dTTP Af%r incubation at 37O for the number of minutes indi(1.7 x log cpm?imole). cated by the nunbers on the curves, reactions were stopped with 20 ,,I of 10 mM EDTA, 1.8 M KOH and loaded on a 5 ml alkaline sucrose gradient (17). Afte-r centrifuging for 17 hr at loo and 36,000 r.p.m. in an M.S.E. SW-40 rotor, 7 drop fractions were collected and acid-insoluble radioactivity (A) or total DNA (B; The pellet ("fraction 0") contained almost all the DNA in ref. 18) measured. the zero-time sample. 256

Vol. 39,No.2,

(Fig. out

IA).

primer

that

la).

its

high

nuclei

assayed

with

of label

phosphates

dGTP and dTTP,

and at least

iiearest-neignbor

analysis

possible

primer increases

were: ApA, 3.033 O.G38 to 3.X6 would

give

(13).

as the only

substrate,

the

in Fig.

resu?ts

significantly cate

little

activated

if

former

for

results

nicks

araCTP.

tor

arabinosyl

residues bacterial

with

DfJase, and hydrolysis

attacilment

to all

as sole

labeled

of endogenous

sequences TpT,

(cf.

ref.

1)

deoxyribonucleotides

nucleotides.

endogenous

4

substrate

to U.051;

transferase

single

is

dCTP,

nucleotide.

adjacent

GpG, 0.037

terminal

synthesis

dATP,

to

of homodinucleotide

to 0.383;

that

from

triphospnate

frequencies

1.3 with

tri-

Di;A could all

the corresponding

labeled

incorpor-

unlabeled

endogenous

however,

nucleotides

The nearest-neighbor

primer

is extensive

from

contribute

analyses

due to terminal

therefore

indi-

transferase.

In the presence

tenini.

incorporation

consistent

inhibitor

cytosine

with

of dTMP (Fig.

a model

DtiA and provides

DTiA synthesis.

Further

to terminal

(cf.

15) sources.

support

iiuclei incorto

of added

IC),

d,lTP,

well

beyond

a Ca 2+ -dependent

endo-

terminator

3) showed

that

label

ed nuclei

(Fig.

28;

(15).

on D!aA with

polymerase

did not label after partial

Centrifugation time)

to nuclear

to "titration"

of from

product

(Fig.

(14)

nuclei by brief

that

and to a expos-

extent

primer

sucrose

inhibiaraCVP

animal

DiJA to a limited

between

studies

the growth

transferring

unactivated activation

in an alkaline intermediate

to account

came from

of a17 available

and the smaller

257

termini

model

terminator,

was in DNA of a size zero

this

a metabolite

to Ca*+ , araCMP was attached corresponding

for

is

u-32P-AraCTP above background, ':ut

lD), probably

3'-hydroxyl

chain

an effective

positions

in which

enough

of DNA synthesis

and is

mainly significantly

chain

tire

unlikely

available

the nuclear This

ure of nuclei (Fig.

of tne three

and

DNA nicking during brief exposure to Ca 2t (Fig. lC), 32 P-dTTP alone, up to a limit probably corresponding

are

the observed

with

level

there

of label

limit.

These nuclease

is

in

covalent

absence

any of the

at all

dCTP and dGTP, the

IJith

in the

from

dTrlP attachment

shorted

approaching

by limited 32P-dT?lP,

incorporation

Labeled

araCTP

Template-independent

it

with-

during isolation 2+ and Ilg inediate

by pancreatic

CpC, 0.3Gg

lC,

initiated

DiM.

?5% of the a- 32P label

855 from

to DirA synthesis.

that

porate

products

(13).

frequencies

bulk

Gmission

in nearest-neigtibor to O.iI81;

and that

2-3 fold.

at least (12)

termini

13).

rates

to acid-soluble showed

23)

triphosphates

(Fig.

labeling

(11)

was immediately

the median

deoxyribonucleoside

5' nucleotides

COMMUNICATIONS

to 1 ml? CaC12, 5 rnli QC12

was observed

apparent

(Fig.

2A) than

exposed

decreased

be hydrolyzed

DMA synthesis

value

(Fig.

were

ANDBIOPHYSICALRESEARCH

Centrifugation in an alkaline sucrose gradient 2+ of Ca continuous nicking decreased the size of

initial

DliA smaller

Mien ation

(Fig.

in the presence

UI~A from

was into then

BIOCHEMICAL

When CaC12 was added,

added

showed the

1970

gradient from

3) resulting

termini (Fig. unactivatfrom

Vol. 39, No. 2, 1970

BIOCHEMICAL

AND BlOPHYSlCAL RESEARCH COMMUNICATIONS

3

6 Fraction

9

12

Number

Figure 3. Activated nuclei (7.9 x lO'/assay) incubated for 45 min in 100 ~1 assay solution (Fig. 11) with MgC12, EDTA, EGTA, dATP, dGTP, dTTP and 23 u;I a-32P-araCTP (2.0 x 10 cpm/umole). Distributions of radioactivity (A) and DNA (B), after centrifuging for 14 nr in the alkaline su rose gradient, were measured as in Fig. 2: +, incubation with no added Ca ii+ ; Q=, with 1 mH CaC12.

extensive

labeling

in

the presence

each case incorporation primer

sites by nicking of nuclear Our experiments do not prove

is

involved

that

this

in physiological ef$ect

DNA synthesis DNA replication

must

--in vivo (16).

of U- 32P-araCTP

of araCMP is a measure

and CaC12 (Figs. 1B & D). In 2+ of Ca -dependent production of

DNA. that the Ca2' -dependent

initiation

be considered and in particular

of DNA replication, in any search for

H.M.,

Progr.

Nucleic~Acid

Res. w.

258

for

N.,

but

factors

endonucleases

The authors acknowledge the technical assistance Mr Mario Froscio and the provision of arabinosyl References 1. Keir,

priming

that

of nuclear they

that

do indicate

control

nuclear

may be involved

of Mrs Donna Staunton cytosine by Dr. W.J.

-,4

DNA

81 (1965).

and Hechter.

in

BIOCHEMICAL

Vol. 39, No. 2, 1970

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Biophys., 133, 238 (1369). Hawk, R., and Wang, T.Y , Arch. Biochem. Biochem. Bi Res. Comn., 21, 235 Brewer, E.N., and Rusch, H, ichim, Siophys. AZ 16T; 455 f D.L., and Muel 'ler, G.m 43: Friedman, 28,(1969). -DeBellis, R.H. ,, Federat ;i on Proc., 2: Thompson, L.R. ,, and Carthy,B.J.TB iochem. Bioohvs. Fes. Comm.. 30. E.A., h,wanJ-?Uiiiiii R.X; 7. Erhan, S., Reisher, S., Franko,

2.

(1965) * 1968). 166 (1968). ilature,

225-, j40 (1970). a. Lagunoff 1 ;. ’ Qf&;e~3E+~ ;o~~9;;) (1969). 9. Kornbero T. . ' . 75.. Science. i54, 1662 (1966). 10. Blobel,-G., an-i, Korib?rg; A., J. Biol. elm., 11. Lehman, I.R., Bessman, M.J.,sS~~S?&d 233, 163 (1958). s., z, 864 (1961). 12. Jesse, J., Kaiser, P.D., and Kornberg, R., J -. -Biol. 13. Waqar, N.A., Burgoyne, and Ptkinson ? R L.A., , '/. ., unpublished results. 14. Momparler, R.L., Biochem. Bio;hys. p,eS. a., 34-, 465 (1969). M.R., D&tsr, M. ., Kornberg, A., Russell, A.F., and Iloffatt, 15. Atkinson, 16. ;1,~~~l~aD~~e~7C(:g69~Foc 17. Olivera: B.M: 1 and Lehman:

ia.

Burton,

K.,

Biochem.

J.,

I:R:: Proc. 62-, 315 m6).'

259

'iatl &'

Acad m' -*

Sci m' --*

U S ti' &"

64 n' -'

1065 (l969) 1426 (IPG7):