Observations on the regulation of DNA biosynthesis

Vol. 14, No. 6, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OBSERVATIONS ONTHERUUUTION OF DNABIOSYNTHESIS Eugene H. Gogol* and Eugene Rosenberg Department of Bacteriology, University of California Los Angeles 24, California

Received

December

30,

1963

It has been established that bacteria not incorporate

radioactive

in stationary

thymidine into DNA (see Lark, 1963).

Three of the possible explanations for the failure thymidine in stationary

phase have been considered:

DN4 polymeraae, (2) depletion nucleoside triphosphates,

phase do

to incorporate (1) inactive

of one or more of the deoxyribo-

and (3) non-functionality

of DNAas a

primer. Experiments with E. co11 starved of an essential amino acid (Billen, Guttes,

1962) and the slime mold g. polycephalium (Guttes and 1961) were interpreted

to indicate

that factors

other than

the enzyme and substrate were responsible for the regulation DNA synthesis. ability

This commmicatiou reports on the relative

of DNA in sonicatee of stationary

to funotion as an active

of in-

phase Pfyxococcus xanthus

primer for DNAsynthesis.

MAUTBRIALS AND MTBODS. 5. xanthus** were grown 2% casitone (generation t5me, 5 hr.),

at 30°C in aerated

harvested by centrifugation,

washed in 0.01 H phosphate buffer, to give a final

0.D.560 ~

pH 7.0, and resuspended in buffer of 20 (20 x 109 cells/ml). Sonicates from

log phase were obtained from cultures which had reached an 0.D.

560 qu

*Participant in the NSFUndergraduate Saience Education Program #E3/3/43-1233. t*A culture of strain PB of 5 xauthus was kindly supplied by M. Dworkiu. 565

Vol. 14, No. 6, 1964 of

0.9.

BIOCHEMICAL

gonicatee

whose

from

etatfouary

0.1). had remained

(final

phase

constant

for

were

RESEARCH COMMUNICATIONS

obtained

at least

from

cultures

two generation

times

O.D. = 2.5). Ten ml suspensions

node1

g-75

(final

gonlfier

by the conversion nucleotides. (one-tenth

log

Analyses scale)

DNA and KNA (Diache,

1955)

DNA* was isolated

the method

of Harmur

by the method

(1961).

of Kirby

RESULTS AND DISCUSSION. stationary

phase

dGTP,

TTP,

and houologous

crude

DNA polyuerase

DNA polymerase

EJ xanthus

of g.

the radioactivity

1 demonstrates

effective

than

the

of the curve,

slope

stjmulated there

sonicate

to the exteni were

50-60

was determined soluble

(Lmry,

log

phase

was a gift

cells

of Dr.

J.

by added

like

1958).

Ninety-five

insoluble

In these

product

homologous

DNA in stimulating

of approximately cpm incorporated

CG

eubtllis

P&e. of dATP,

respects,

the well

the studied

percent

was solubilited

of by

DNA was more From

DNA synthesis 8 cpm/pg

with

added

no added

and had a M.U.

566

Kg++,

DNA synthesis.

may be seen thet

69%

to

of DNA by sonlcates

behaved

+The & m DNA contained 2 x 106 (unpublished results).

1951).

from Bacillus

xanthus

added

of

according

1).

that

oligo-

et al.,

DNA (Table

(Lehman,

it

of the

the determination

was stimulated

of the acid

DNAase. Fig.

for

The synthesis

of 5

cells.

KNA, extracted

(1956),

demonstrated

out by micrasodifications

methods

from

30 seconds

a modification

and protein

k& xanthus

for

DNA to acid

carried

of existing

probe

DNAase activity

insoluble

were

in a Brauson

examinations

using

procedure.

of acid

broken

and stationary

was assayed

(1958)

inch

Microscopic

in both

DNA polymerase

were

a one-half

5°C).

brealcage

Lehman et al.

of the cells

with

temperature

complete

Since

AND BIOPHYSICAL

was DNA.

DNA, one

of approximately

Vol. 14, No. 6, 1964

BIOCHEMICAL

AND BIOPI-IYSICAL RESEARCH COMMUNICATIONS

Table 1 Requirements for DNASynthesis

Complete system*

Incorporation of dATP-8-C14 (cpm) 155*

minus TTP minus dGTP

71 28

minus dCTP minus TTP, dGTP, dCTP minus Mg++ minus added DNA minus added DNA+ 9 ug RNA

28 10 5

Components

87 a9

Womplete system contains glycine buffer, pR 9.0, 20 um; MgC12, 2 p; P-mercaptoethanol, 0.3 um; TTP, dATP, dGTP, and dCTP each 5 uum; DNA, 9 ug; dATP-8-C", 5.65 p&m, 16,000 cpm; and stationary sonicate, 0.15 ml (53 mg protein/ml), all in 0.3 ml; incubation time 15 minutes at 37oc. *Incorporation at zero tfrPe has been subtracted,

0

I 0

I 4

I Added

I 8 DNA t p 9)

I

I 12

I

Fig. 1. Stimulation of DNAsynthesis by added DNA. The assay system is identical to that described in Table 1 except that the quantities of added DNAwere varied. The curve represents experiments done on stationary aonicates prepared at 3 different times, all adjusted to contain 7Oug sonicate DNA. 567

I 16

Vol. 14, No. 6, 1964 would

predict

stationary that

that

the sonicates

regard that

than

stationary

Fig. (1:4

approximately

However,

chemical

contained

to priming log

analysis

70 2 3 pg DNA.

activity.

phase

Preliminary

sonicate

phase

the specific

DNA is

sonicate that

RESEARCH COMMUNICATIONS

7 ug DNA in

DNA is approximately

2 demonstrates

dilution),

AND BIOPHYSKAL

were

sonlcate

revealed the

there

sonicates.

the stationary with

BIOCHEMICAL

This

the

demonstrated suggests

that

902 nonfunctional experiments

a more active

have

primer

DNA.

at a concentration activities

of 3 mg protein/ml

of DNA Polymerase

of the

TIME (MIN)

of DNA synthesis by sonicates of stationary and Fig. 2. The kinetics The assay system was as described in Table 1 log phase g. xanthus. except that excess salmon sperm DNA (32 pg) and ATP (0.5 ~1111)were Multiple amounts were used and 0.5 ml aliquote were withdrawa added. Dilutions of the sonicates were msde with .Ol H at various times. phosphate buffer. 568

BIOCHEMICAL

Vol. 14, No. 6, 1964 stationary

and log

phase

failure

of the resting

simply

to the absence Increasing

incubation

result

from

from

to synthesize

the

DNA can not be attributed

of log

phase

sonicates

3 to 12 mg protein/ml

in the rate

result

of DNA synthesis

in the in approximately

(Fig.

2).

However, sonicates

in the concentration

of stationary

phase

in approximately

a twofold

in rate.

In similar

it

was found

with

that

increasing

increased

protein

Therefore,

identical.

of DNA polymerase.

decrease

of dATP-8-C 14 incorporation

the rate

concentrations

O-24 mg protein/ml,

activity

are

increases

experiments increased

cells

increase

identical

sonicates

the concentration

mixtures

a threefold

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

while

from

of log

in stationary

phase

phase

O-3 mg/ml and then

sonicates

sonicates,

decreased

the

at higher

concentrations. The inhibition

of DNA polymerase

trations

could

activity

of DNAase in log phase

stationary

at the higher

not be due to DNAase activity

phase

soricates

sonicates

under

protein

since is

the conditions

the

slightly

concen-

specific higher

used

than

to assay

for

the DNA polymerase. In conclusion of n. xanthus (1) measured (2) which

is (3)

activity

it

has been

that

stationary

sonicates

contain:

As active

a DNA polymerase

at low protein an inhibitor not expressed DNA which for

observed

as log

phase

sonicates

if

concentrations, for

the DNA polymerase

in log

phase

sonicates,

is 90% nonfunctional

with

system

(not

DNAase)

and respect

to priming

DNA polymerase.

Billen, D., Biochem. Biophys. Res. Ccuss. 1, 173 (1962). Dische, Z., in E. Chargaff and J. N. Davidson, The Nucleic -p-t Vol. 1, Academic Press, New York, 1955, p. 285. 569

Acids

Vol. 14, No. 6, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Guttes, E. and Guttes, S., Proc. Am. Sot. Cell Biol., p. 79 (1961). 405 (1956). Kirby, K. S., Biochem. J. 3 Genetics, Lark, K. G., in J. Ii. Taylor, Progress &Molecular Part I, Academic Press, New York, 1963, p. 153. Lehman, I. R., Bessman, H. J., Sims, E. S., and Kornberg, A., J. Biol. Chem. 233, 163 (1958). Lowry, 0. H., Rosanbrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chew, l!I3, 265 (1951). Marmur, J., J. Mol. Biol. 3 208 (1961).

570