Alkali-labile colicinogenic factor E1 DNA molecules formed in the presence of N-methyl-N′-nitro-N-nitrosoguanidine

BIOCHEMICAL

Vol. 74, No. 2, 1977

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

COLICINOGENIC FACTOR El DNA MOLECULES FORMED IN THE PRESENCE OF

ALKALI-LABILE

N-METHYL-N'-NITRO-N-NITROSOGUANIDINE H. Mizusawa,

S. Tanaka,

M. Kobayashi

and K. Koike

Cancer Institute (Japanese Foundation Toshima-ku, Tokyo 170, Japan

Received

November

for

Cancer

Research),

Kami-Ikebukuro

23,1976

SUMMARY Plasmid ColEl DNA of E. cofi was used as a target DNA molecule to analyse the structural modification of DNA by N-methyl-N'-nitro-N-nitrosoguanidine. When the low concentration of this drug was used, both cell growth and overall DNA synthesis were neither stimulated nor inhibited, and plasmid DNA molecules were isolated as closed circles after replication. These molecules were stable for the ribonuclease treatment, but became susceptible to the alkaline hydrolysis. Such alkali-labile sites of ColEl DNA were found in the parental strands and randomly distributed from the restriction endonuclease EcoRl cleavage site. INTRODUCTION N-Methyl-N'-nitro-N-nitrosoguanidine(MNNG) which

carcinogen, cells

the mutagenic

observed if

the

at low

the

in the

action

concentration

of

(4),and

As it

intermediary

MNNG is

directly

was used ColEl

system.

as a target

0

1977

by Academic in any

of reproduction

also

extensively

relaxation where

inhibited

daltons

Press,

form

(7).

Inc. reserved.

such

supercoiled these

570

were

reported

cell,

site(s) the

and found the

overall

even

MNNG acts

alkali-labile

effect that

of the

closed-circular activity

of

modification

or carcinogenesis,

to analyse

With

frequently

studied

of

bacterial

were

animal

the

structural

mutation

molecule

the

In the

or

low rep-

DNA syn-

(6).

the

DNA is a double-stranded

weight of 4.2~10~

On the

to create

the

whether

to the

mutations

observations

known

mutagen

DNA(l).

of mtDNA replication

about

nor

ambiguous relating

is

cleavage,

stimulated

still

Similar

steps

of MNNG brought

is

and double

We have

DNA (5).

was neither

evident,

chromosomal

MNNG was used(3).

DNA by single-strand

thesis

the

paint(2).

carcinogen

MNNGon various

Copyright All rights

is

replication

chromosomal

licating

by methylating

concentration

as a powerful

cu.&

acts

is a well-known

of ColEl

correlation circle

characteristics,

DNA of

in the with

DNA by E.

simple

a molecular

one can easily

de-

Vol. 74,No.

tect

a single-stranded

ColEl

DNA replication

(8),

and the

DNA is

of

AND BIOPHYSICAL

the

DNA.

has been studied

a useful

by chemical

Furthermore,

in detail

experiment for

analysing

we present

our

findings

the

using

has been also

material

RESEARCH COMMUNICATIONS

cell

mechanism lysate

of E. co&

established(g).

a mechanism

of

ColEl

of mutation

induced

carcinogen.

In this

paper,

ccDNA)

li-labile, rental

cleavage

transformation

thus,

(ColEl

BIOCHEMICAL

2, 1977

formed

in the

presence

and such alkali-labile strands

of the

of sites

that

the

low concentration were

randomly

closed-circular of

ColEl

DNA

MNNG became alka-

destributed

in the

pa-

molecule.

MATERIALS AND METHODS ColEl'), kindly 1. T3achhh.t ~tin and medium - E. euti K12 A745(met,thy, supplied by Dr. Sakakibara, National Institute of Health, Tokyo, was cultivated in the OC medium containing 4 pg/ml thymine with shaking at 37"C(8). The growth was monitored with absorbance at 660 nm. DNA was labeled with [3H]thymine(10 Ci/mmole, NEN Corp.) or [14C]thymine(51 mCi/mmole). The doubling time of the log culture was about 30 min, under the conditions used. 2. PhepahcLtion 06 doned-CirrclLeah CoLEI VNAColEl ccDNA was prepared by the method of Clewel and Helinski (1O)with slight modification. Cleared lysate, prepared from 1010 cells, was treated with 1 %SDS and extracted with saturated phenol, followed by dialysis against TES buffer(O.O5MTris-HCl,pH8.0, 0.05 M EDTA and 0.5MNaCl). The dialysed sample was subjected to the equilibrium centrifugation in CsCl of 2.79 and 6OOpg of ethidium bromide were disCsCl-ethidium bromide. solved in 3ml of the DNA solution. The centrifugation was performed in a Beckman SW50.1 rotor at 33,000 rpm for 40 hr at 20°C. After centrifugation, drops were collected from the bottom of the tube and 5~1 aliquote of each fraction was applied onto a filter paper disk(24mm, Toyo No.514), and washed twice with 5 % trichloroacetic acid(TCA). Filter paper disks were counted in a Beckman LS-250 liquid scintillation counter. Then, the ccDNA fractions were collected, and ethidium bromide was eliminated by tert-butanol, followed by dialysis against TES buffer.

3. NeLLthd and &kaLLne &~c~~obe detiity g&a&e& cenXhi~~~gaLion -About 200 ~1 of DNA sample was layered on the 4.5 ml neutral sucrose gradient(5-20%)in O.O25MTris-HCl buffer, pH8.0,containing O.OlM EDTA and 0.5M NaCl (10). Centrifugation was carried out in a Beckman SW50.1 rotor at 45,000 rpm for indicated time at 5°C. The tube was, then, punctured and drops were directly colThe radioactivity was measured, as described lected on filter paper disks. above. In the case of alkaline sucrose density gradient centrifugation, 0.6 ml of 60 % sucrose was placed in the bottom as a cushion, and sucrose gradient of 5-20 % in 0.26 M NaOH was made. The alkali-treated sample was layered on the gradient and centrifugation was performed at 45,000 rpm for 5 hr at 5°C. RESULTS

1. Fahmtian Growth labeled

of

C&El. VNA cell and the

06

the

thymine

in the

in

Rhc

phc%nnce

overall

presence

synthesis of

lug/ml 571

04

Low

concenXha;tion 06 MNNG -

of DNA observed MNNG are summarized

by the

uptake

in Fig.l.

of Under

Vol. 74, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0 30 60 -60 -30 TIME IN MINUTES Fig. 1. Effect of MNNG on the cultivation of E. coti. The cells were grown in the OC medium containing [14C]thymine(0.5$i/ml)with shaking at 37°C. At 0.1 of absorbance at 660 nm, cells were harvested, washed once with glucoseand thymine-free OC medium and suspended in the fresh OC medium containing [3H]thymine(0.5pCi/ml), followed by incubation. After 5 min, MNNG(l pg/ml)was added to a half of the culture. The rest was the control without MNNG. From these cultures, 0.1 ml aliquotes were taken at the indicated times and mixed with cold 5 % TCA. The precipitate was collected by Whatman GF/C(25mm) filter and washed with cold 5 % TCA and with ethanol. Filters were counted in a toluene based scintillation fluid. Absorbance at 660 nm with(I--@) or without (x--x) MNNG. Incorporation of [14C]thymine with (M) or without (w)MNNG. Incorporation of [3H]thymine with (A-4) or without (o-o) MNNG.

the tion

conditions of

described

[3H]thymine

same as those the overall These

into the

control.

legend,

cell

with

both

in the

This are

are consistent

not

presence

indicates affected

the

growth

the

cell

and incorpora-

of MNNG were

that by this

previous

of

the

growth

almost

of

the

concentration

results

reported

the cell

and

of MNNG. by Sanchetzs

and

(3).

As depicted rectly

in the

subjected

of

unaffected

[3H]

legend

to equilibrium

The radioactivity ratio

the

DNA synthesis

data

Ormedo

of

in the

of to

[14C]

by lug/ml

the

of Fig.2,

pronase-treated

centrifugation

closed-circular is

shown in

cell

in CsCl-ethidium DNA fraction

Fig.2.

of MNNG, although

Synthesis there

572

lysate

bromide

was measured of

was a little

ColEl delay

was di(11).

and the

ccDNA was almost in its

synthe-

BIOCHEMICAL

Vol. 74, No. 2, 1977

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

30 60 TIME

IN MINUTES

Fig. 2. Effect of MNNG on the ColEl DNA synthesis. From the culture, as described in Fig. 1, 10 ml aliquotes were taken at 10, 30 and 60 min, and DNA synthesis was terminated by 20 mM KCN. Cells were precipitated, washed with 0.8 % NaCl in 0.2MTris-HCl, pH8.0and resuspended in 1.5ml of O.O2MTris-HCl, pH8.5,0.025M EDTA. The suspension was,then, incubated at 0°C for 20 min with 0.15 ml of lysozyme(4mg/ml). The lysozyme-treated cell was mixed with1.5 ml of 0.75 % sarkosyl and lysed at 65°C for 8 min. Pronase E(2mg/ml) of 0.15 ml was added to the lysate and incubated for 60 min at 37°C. This pronasetreated lysate was vigorously mixed with vortex mixer, then centrifuged in After centrifu ation, CsCl-ethidium bromide (11). the radioactivity of ccDNA fraction was measured. Ratios of [3H] to [ 9 4C] were plotted. MNNG added (bn) and the control without MNNG (O-O).

sis.

This

slightly

may indicate different

from

2. Aeha.kX-tib&a As it

is

parental cell,

interesting strand

that

of

with

WE1

DNA domed in the ptience

o6 MNNG-

with

60 min.

final

in both

dients.

a single

in Fig,3a,

modification

washed thymine-free

concentration

The ColEl

DNA.

experiments

in the

AND METHODS, was centrifuged As shown

to MNNG is

[3H]thymine(5pCi/ml),was

culture

further

DNA synthesis

a structural

DNA, following

and resuspended

to the

of ColEl

the chromosomal

to know whether of ColEl

labeled

nued for

a sensitivity

c&bed-chcuhk

xlO8cells/ml) added

that

ccDNA,

of prepared

neutral peak of

were

carried

once

at mid-log

OC medium. 1 pg/ml

at the

out.

The phase(2-4

MNNG was,

and incubation

as described

and alkaline the

occurs

conti-

in MATERIALS

sucrose

ccDNA(23s),

then,

which

density

gra-

was stable

Vol. 74, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICALRESEARCH

FRACTION

COMMUNICATIONS

NUMBER

Fig. 3. Neutral and alkaline sucrose gradient centrifugation of MNNG-treated ColEl ccDNA. [3H]Thymine-labeled cells were harvested, as described in the RESULTS. The cleared lysate was prepared according to the legend of Fig. 2, and MNNG-treated ColEl ccDNA was extracted as shown in MATERIALS AND METHODS. (a) MNNG-treated [~HIccDNA was centrifuged in 5-20 % neutral sucrose gradient at 45,000 rpm, for 180 min, at ~"C(O-O). (b) MNNG-treated [~HIccDNA was dialysed against pH12.0for 180 min, at 37°C and then subjected to 5-20 % alkaline sucrose density gradient centrifugation(M),as described in MA ERIALS AND METHODS. As the internal marker, the ribonucleotide-containing [ 14 C]ColEl ccDNA(8)was added. Peaks at 17th and 20th fractions corresponding to singlestranded circular(l9 s) and linear(l7 s) DNAs, respectively(0--0). MNNG-treated [3H]ccDNA in 0.1 MTris-HCl,pH 7.6, containing 0.5mMEDTA and 10mMMgC12 was digested with restriction endonuclease EcoRl for 30 min at 37"C(12). (c) EcoRl-digested DNA(o-o)was centrifuged in 5-20 % neutral sucrose density gradient with the 23 s internal marker of [14C]ColEl ccDNA(M),separately prepared in the control culture. (d) EcoRl-treated DNA was dialyzed against alkali and centrifuged in 5-20 % alkaline sucrose density gradient(M) . As the 17 s internal marker, EcoRl-digested [14C]ColEl DNA was added (o-o).

for

the

ribonuclease

the

neutral

the

radioactivity

sedimenting

treatment(1

sucrose

peak(55

slowly-sedimenting sized

ColEl

ed circular the

total

While

gradient. was split

into

s) was the peak(17

mg/ml

at in the

DNA(19

The slowly-sedimenting the

conditions

at

used.

574

density

(Fig.3b).

linear

the

position peak

On the

ColEl form

of the

in Fig.Sb

other

in

gradient,

The rapidly-

MNNG-treated

s) was a single-stranded shoulder

was observed

sucrose

peaks

alkali-resistant

a small

DNA, under

60 min),

alkaline

two distinct

DNA, having s).

37°C for

hand,

of

DNA. the

The full-

single-strand-

was about

50%of

the

ccDNA

ColEl

Vol. 74,No.

isolated

2, 1977

from

condition

BIOCHEMICAL

the

(data

control not

ccDNA molecules,

E. co.&

internal

sucrose

peak of

the

DNA became After

density

EcoRl

DNA from

in Fig.3d, tions,

the

fairly

Total

counts

[3H]DNA. Fig.3b.

the in the amount

Results

in

distributed

from

the

observed

molecules

culture

lower

the

were

EcoRl

in Fig.3c,

which

is

by alkaline

As shown

were

about

50 % of the

sites

ColEl

DNA in

are randomly

a small

corresponding EcoRl

frac-

as a 17 s peak.

alkali-labile

to the

[14C]

molecular-weight

In addition,

may be resistant

sucrose

marker.

linear

possibly

with

MNNG-treated

single-stranded

site.

and

As shown in

internal

lower

used

EcoRl

EcoRl-digested

fractions

the

EcoRl

digestion.

DNA was observed

to the

with

that

EcoRl

as the

to the

that

cushion

indicating

experiment,

linear

restriction

the

ColEl

17 s as compared

DNA was analysed

tailed

indicate

at

by the

was added

is comparable

at

form

molecular-weight

Fig.3d

the

DNA(12),ccDNA,

centrifugation.

s),

In this

single-stranded

This

[3H]DNA

ccDNA(23 linear

many counts

although

of

endonuclease

ColEl

DNA was found

MNNG-treated

control

alkaline

of MNNG, became

digested

gradient

[~H]co~E~

centrifugation.

in the

was completely

density

[14C]ColEl

digestion,

a part

restriction

site

Fig.3a,

double-stranded

gradient

ColEl

of

Since

one specific

neutral

marker

that

of low concentration

h&&t-

of

main

to this

strands.

experiment

to the the

only

RESEARCH COMMUNICATIONS

resistant

indicates

presence

parental

cleaves

previous

Fig.3c,

ColEl

clearly

va the &ha,&-labtic

subjected

the

was completely

This

in the

on their

3. Pohtion

in the

shown).

formed

alkali-labile

from

culture

AND BIOPHYSICAL

to

amount the

of

23 s [3H]

digestion.

DISCUSSION From the ates

results

a structural

ing

in the

from

the

described

modification

formation

restriction

methylate

the

N-3 position

in the

chromosomal

ColEl

it

on the

site

DNA(13),it

DNA molecule

is

is

likely

parental

of alkali-labile

EcoRl

the

above,

strand which

sites, in the

molecule.

of adenine

and the

is easily methylated

of ColEl

are Since

that

presence

that

randomly

distributed

MNNG is well-known

some purine of

MNNG cre-

ccDNA, result-

N-7 and O-6 positions

expected

in the

indicated

to

of guanine base(s)

low concentration

in of

Vol. 74, No. 2, 1977

BIOCHEMICAL

MNNG, and an alkali-labile tion not

of methylated observed

vation

is distinctly

in V&O

replication

prelabeled

parental

tion

reaction

tion.

This

experiments sults

will

the

synthesis

is

apurinic

DNA(13).

in the

site

However,

cleared

lysate

different

strand

from

of

ColEl

of DNA is

event

elsewhere.

is

and not

site suggested

dependent

for

interesting

to

know a repair

to analyse

the

of

replication

the

Kimball fixation

mechanism of

studies

experiments

Recently

the

shown).

of mtDNA exposed

it

by depurina-

open-circular

not

alkali-labile

and inhibitor

essential

(data

by preliminary

therefore,

product

case

formed

of the

case

ccDNA,

is examined

bromouracil

be published

the

RESEARCH COMMUNICATIONS

subsequently

an increase

As the

a premutational

possibility

is

in this

system(6).

using

AND BIOPHYSICAL

This

obser-

to MNNG in the was formed

that on the

on the

such modificaDNA replica-

on density-shift by nalidixic

and Setlow of mutation(14). of alkali-labile

alkali-labile

DNA was

ColEl

acid. indicated It

Rethat

is,

ccDNA,

and

DNA as a tem-

plate. ACKNOWLEDGEMENT We thank Dr. H. Sugano, Cancer Institute, for his interest and encourageThis work was partly supported by a Grant-in-Aid for Cancer Research ment. from the Ministry of Education, Science and Culture, Japan. REFERENCES 1. Siissmuth, R., Haerlin, R. and Lingens, F. (1972) Biochim. Biophys. Acta 269, 276-286 2. Cerdd-Olmedo, E., Hanawalt, P.C. and Guerola, N. (1968) J. Mol. Biol., 33, 705-719 Jimenez-Sanchez, A. and Cerdd-Olmedo, E. (1975) Mut. Res., 28, 337-345 Swann, P.F. and Magee, P.N. (1968) Biochem. J., 110, 39-47 Olson, A.O. and Baird, K.M. (1969) Biochim. Biophys. Acta, 179, 513-514 Koike, K., Kobayashi, M., Fujisawa, T. and Tanaka, S. (1975) Biochim. Biophys. Acta, 402, 352-362 7. Bazaral, M. and Helinski, D.R. (1968) J. Mol. Biol. 36, 185-194 8. Sakakibara, Y. and Tomizawa, J. (1975) Proc. Natl. Acad. Sci., 71, 802-806 M. and Higa, A. (1970) J. Mol. Biol. 53, 159-162 9. Mandel, 10. Clewell, D.B. and Helinski, D.R. (1970) Biochemistry, 9, 4428-4440 11. Takeda, Y., Matsubara, K. and Ogata, K. (1975) Virology, 65, 374-384 12. Inselburg, T. (1974) Proc. Natl. Acad. Sci., 71, 2256-2259 13. Lawley, P.A. and Orr, D.J. (1970) Chem.-Biol. Interactions, 2, 154-157 14. Kimball, R.F. and Setlow, J.K. (1974) Mut. Res.,22, l-4

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