Evidence for the synchronous replication of mitochondrial DNA during the yeast cell cycle

BIOCHEMICAL

Vol. 101, No. 4,19Bl August

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 1350-1356

31, 1981

EVIDENCE FOR THE SYNCHRONOUS REPLICATION

OF MITOCHONDRIAL

DNA DURING THE YEAST CELL CYCLE Stephen

F. Cottrell

Department

of Biology,

The City

July

13,

Brooklyn

University

Brooklyn, Received

and Lee H. Lee

of

New York

College

of

York

New

11210

1981

SUMMARY

The nature of mitochondrial DNA replication during the synchronous cell cycle in the yeast, Saccharomyces cerevisiae has been investigated by examining the rate of labeled DNA precursor incorporation into specific segments of the mitochondrial genome at defined points during synchronous growth. The movement of label uptake from one area of the DNA to another at different times during the synchronous cell cycle indicates mitochondrial DNA replication to be a synchronous process during this time with most or all molecules at the same point in replication at any given time during the cell cycle. INTRODUCTION The synthesis throughout time,

the entire

however,

remains

of mitochondrial

the exact

unclear.

molecules

(there

at random

molecules

could

replication points

are

directions might

ndture

cell,

the

synchronously fork(s)

each molecule.

Still

population

Inc. reserved.

cycle

50 molecules initiate or,

from moving other

1350

a specific

At this

process of these per haploid

and complete alternatively,

initiation

possibilities

replicaall

in the cell

in the same direction more exotic

place

(l-3).

replication

at the same point

be envisioned.

,Z 1981 b.v .4cademic Press, of reproducrion tn any form

could

to take

cerevisiae

intracellular

4,5)

0006-291X/81/161350-07$01.00/0 Copyrighl .4ff rights

5.

continuous

the cell

replication

the replication in

of this

throughout

initiate

the yeast,

is known

to be approximately

per diploid points

in

one hand,

believed

proceeding with

cycle

On the

and 100 molecules tion

cell

DNA (mtDNA)

mtDNA

cycle

point

or

or also

with

Vol. 101, No. 4,198l

BIOCHEMICAL

In the present tion

during

study

the yeast

precursors and following

label

genome after

cycle

administration

label

incorporation

into

synchronous ous cell

This

with

in this

areas

uptake

into

procedure

specific

endonucleases

us to distinguish during

of

isolated and assessing

separated

modes of mtDNA replication

cycle

segments

mtDNA molecules

restriction

has allowed

cell

DNA

of the mitochondrial

each of the electrophoretically

and asynchronous cycle

of labeled

synchronous

different

by dissecting

of mtDNA replica-

pulses

a single

Label

was determined

each pulse

fragments.

into

period.

after

tion

during

incorporation

RESEARCH COMMUNICATIONS

the mechanism

by administering

intervals

each labeling

the mtDNA molecule

BIOPHYSICAL

we have analyzed

cell

at different

AND

restric-

between the

the

synchron-

yeast. MATERIALS

AND METHODS

A diploid strain of 2. cerevisiae (ATCC 42029) was employed in this Both cells svnchronized by the modified (6) selection and induction study. method of Williamson-and Scopes (7) as well as asynchronous control cells were separately inoculated at initial cell densities of 2 g wet weight/ml in synthetic medium (8) and grown with vigorous aeration at 25". The quality of cell synchrony was determined by monitoring cell morphology and total cell At specific periods during a single synchronous cell cycle DNA content (9). or during exponential growth in the case of the asynchronous cells 160 ml aliquots of culture were removed and incubated for 15 min periods with 6 New York). uCi/ml of (2-3H) adenine (28 Ci/mmole, Schwarz/Mann, Orangeburg, Label uptake was terminated by the addition of KCN (1 me/ml), and immediately followed by 2 washes in chilled distilled H20 containing 1 mgfml KCN. All cell pellets were stored on ice until the completion of the sampling period. After the addition of cold carrier cells to each labeled sample, mitochondria were isolated according to the snail gut enzyme procedure of Casey --et al. (10) The mitochondrial pellets were with 1 mg/ml KCN included in all solutions. After resuspended in 10 mM Tris, pH 7.4, 2 mM EDTA and lysed in 1% sarkosyl. mtDNA was isolated by preparative an initial deproteinization step (ll), the gradients fractionated and CsCl density gradient centrifugation (12), extensive the mtDNA located by determining absorbance at 260 nm. After dialysis against 10 mM Tris, 2 mM EDTA at pH 7.4 in the cold, the mtDNA was concentrated by ethanol precipitation and subjected to either Barn HI or Hpa I digestion according to the manufacturer's instructions (New England Digested DNAs were fractionated by electroBiolabs, Beverly, Mass.). phoresis (13), the gels sliced, the gel slices (1 mm thick) solubilized (14) and counted in a toluene-based scintillation fluid (12). RESULTS AND DISCUSSION By following chondrial

label

incorporation

genome at different

into

intervals

various

throughout

1351

fragments the

of the mito-

synchronous

cell

cycle

BIOCHEMICAL

Vol. 101, No. 4,198l

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

2

1

3

p 40

a

5 z 20 e 8 .c 0 01 E 3 40 8 ml 20 a 5 0

4 3

20-

f m k E2

IO8 6-

-i

4-

k P

20-Q

2 0

z

E 40 z k P 20

t

01

1

2

5

0

20 30 10 gel slice number

1.

Times of (3H)adenine pulse label administration chronous growth are designated by the arrows. typical yeast cell profiles at 3 times during growth are also given.

Fig.

2.

The percent of total counts incorporated into each restriction fragment at the 3 times of pulse label administration during synchronous growth. In this case fragments were generated by Barn HI.

at least

of label

centage nearly

same during

stages

Alternatively,

label

pulse

with

delivery

each fragment cycle

The arrows chronous

cell

the distinct

centered

the percentage

be indicative in Figure

cycle

increases

it

with

of total

mtDNA label

pulse

labeling

those

1352

the per-

be assumed

all

molecules

delivery. after

each

incorporation periods

into

during

the

mode of mtDNA replication.

times

of tritiated in total

found

remaining

could

of each pulse

of a synchronous

1 designate

with

one or a few fragments

different

when each pulse stepwise

periods

If were

any one fragment

was random

at the time

cycle

fragments

labeling

mtDNA molecules

in just

during

in the cell

into

pulse

of replication

shifting

would

different

be anticipated.

restriction

incorporated

of these

at different

all

during synDrawings of synchronous

might

points

essentially

mtDNA label

the replication

of results

at different

into

of total the

two types

delivered

to be incorporated

Both

4

Fig.

pulses

cell

3 (hours)

Time

in -S. cerevisiae

that

02

I

I

during adenine

cell

the

second

syn-

was administered.

DNA content

and the regu-

Vol. 101, No. 4,1981

lar

appearance

achieved

of new buds

during

results the

BIOCHEMICAL

all

digested lyzed

for

Barn HI,

label

detectible labeling

with

the

cycle

ration

uptake.

for

observed synchronous level

of label asynchronous,

label

administration

function which tion

is

into

time

fragment pulse

intervals

labeling

degree conditions

during

yet

another

fragments

the cell remains

during

are

clearly

compatible

of synchronous

cell

complement

with

occurs

(Figure a model

of mtDNA molecules

1353

constant

of pulse

enzyme

a

(Hpa I)

a comparable

situa-

of total

to fragment

with

label at differ-

restriction

at 3 comparable label

uptake

again

at least

some

6). in which

the synthesis

The extent

growth.

the

to be just

5) whereas

growth

during

the

during

periods

constant

size

that

from mtDNA isolated

appears

fragment

relatively

of fragment

evident

the percentage

(Figure

asynchronous

synchrony

at all

our strain,

from

cycle

is

independent

restriction

growth

to change

at each labeling

generated

uptake

with

synchronous

a function

of replicative

intracellular

Using

mtDNA

to the relatively

cells

label

during

of incorpo-

reproducible

contrast

Here

incorporation

just

sharp

growing

observed

periods

Our data

the

During

is highly

4).

5 detectible

mtDNA is

label

apparently

size.

it

of pulse

of total

as 2 other

data

each of the 3

levels

percentage

2 as well

each Barn HI fragment

(Figure

observed.

uptake

in

and ana-

incorporation

fragment

From these

logarithmically

of fragment generates

is

each of

3 periods

in label

the

growth,

into

each of the

was

2 shows

after

incorporation

during

each fragment

into

Figure

electrophoretically

an increase

3.

synchrony

synchronous

A summary of the

This

cycle.

from

separated

in Figure

in

of cell

1 and 3 show decreasing

in Figure

uptake

during

each Barn HI restriction

movement

cell

level

mtDNA was isolated

of label

fragments

reported

given

label

fragments

RESEARCH COMMUNICATIONS

examined.

administration

same period.

the data is

in which

2 exhibiting

into

a high cycles

seen to shift

whereas this

experiments

ent

is

incorporation

period

that

The level

fragment

during

label

the

fragments

cell

label

BIOPHYSICAL

cell

experiment

of pulse

with

indicate

3 synchronous

of a typical

3 periods

AND

of this

remains

of mtDNA under synchrony

unclear

within

at this

time

BIOCHEMICAL

Vol. 101, No, 4,1981

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

5 i?

frag

2

frag

3

frag

1

.5 2’

“1

s 8

40-

mI z

-

.5

fraction

.75

of cell

Label

most

into each Barn HI fragment is plotted against of an asynchronous cell generation when pulse label occurred. This figure represents a summary of 2 experiments.

not

there

into

compatible

replication

in here

at least

the probable

this

yeast

unperturbed,

reflect

exponential

scheme of cell

growth cells. into

cannot

the

true

never

drops

other

cell

this

cellular

cycle.

These

origins

of mtDNA

that

the

data

method

to induce

cell

seems unlikely parameters

used

fragments

1354

during

since

under

have been

of unperturbed,

we have measured

restriction

findings

be excluded

characteristic since

indi-

the mitochon-

mode of mtDNA replication

but

to zero

fragments

within

of multiple

of the particular

(3,6,9,17) Moreover,

of the

fragments

The observation

in yet

of replication

It

16).

numerous

different

increases

existence

growth,

synchrony

balanced growing

(15,

the result

and do not

fragments

some portions

with

are

other

sites

restriction

may be involved.

certain

may be multiple

genome during

among certain

molecules

uptake

drial

incorporation

all

movement

before

that

synchrony

if

to decrease

cates

reported

of label

incorporation

fails

1

incorporated

that

and/or

3

.75

4.

label

frag

generation

Fig.

suggests

tially

.5 of cell

Label incorporated into each Barn HI fragment is plotted against that fraction of the synchronous cell cycle when pulse introduction occurred. The beginning of each cell cycle is defined by the first appearance of new buds and the initiation of stepwise increases in total cell DNA content. This figure represents a summary of 3 independent experiments including the one presented in Fig. 2.

the amplitude

exhibit

.25 fraction

0

;a

3.

although

are

4

v-

Fig.

that fraction introduction independent

that

r z

0

cycle

1

e4
_

T.3

1

frag

20-

2

.25

G

with

alterations time

our

shown to exponenin

and these

label

Vol. 101, No, 4,198l

BIOCHEMICAL

40

30

20

AND

frag

3

frag

2

frag

1

BIOPHYSICAL

RESEARCH COMMUNtCATIONS

b ,I 5 z

-

fw

1

dfrag2

20-

10 1 0

.25

3

fraction

frag frag

.75

of cell

_

5

5 4 1

E 8 5

06

cycle

0

p

.25

.5

fraction

of cell

.75

1

generation

Fig.

5.

Label incorporated into each Hpa I fragment is plotted against that fraction of the synchronous cell cycle when pulse introduction occurred. This figure represents a summary of 2 independent experiments.

Fig.

6.

Label

incorporated into each Hpa I fragment is plotted against fraction of an asynchronous cell generation when pulse label introduction occurred. that

values

are

expressed

that

time

this

molecule

adenine

point,

cell

cycle

has been

nothing

is

because

of the

existence our

enable this

known

obtained

synchrony

label-restriction

strains

reported

the

the

exact

fragments

2. cerevisiae,

appear

have been mapped,

thus

1355

label

during

the

genomic 1lY

organism

intermediates. cycle

dissection

The

in conjunction paradigm

should

of mtDNA replication in the

present

genome of the strain

somewhat

of

essentia

in this

cell

generated

in

here.

mitochondrial

mechanism

extent

total

to occur

replicating

during

in the mitochondrial

fragments that

intact

endonuclease

to examine

The restriction

these

the yeast,

in

at

to occur

the

changes

the mode of mtDNA replication

of replicative

been mapped

the

concerning

from

to isolate

pulse

nor

known

influences

the data

of information

incorporation

heterogeneity

genome reported

affect

inability

yeast.

Moreover,

would

about

us to begin

not yet

other

(3,19)

mtDNA label

clearly

each fragment

the mitochondrial

the bulk

organization

see 18) which

into

into

of total

the base sequence

review

incorporation

Although

with

neither (for

incorporation yeast

as a percentage

different we are not

from yet

those

study

in have

employed. generated

in a position

in to

Vol. 101, No. 4,198l

trace

the

exact

mtDNA replication

8lOCHEMlCAL

movement during

AND

of label the yeast

BIOPHYSICAL

incorporation cell

RESEARCH COMMUNICATIONS

and hence

the mechanism

of

cycle.

ACKNOWLEDGMENTS This study was supported by Public Health Service grant GM25901 from the National Institute of General Medical Sciences and a PSC-CUNY grant from The City University of New York. REFERENCES 1. 2. 3. 4. 5.

Williamson, D.H. and Moustacchi, E. (1971) Biochem. Biophys. Res. Commun. 42, 195-201. Sena, E.P., Welch, J.W., Halvorson, H.O. and Fogel, S. (1975) J. Bacterial. 123, 497-504. Cottrell, S.F. (1981) Exp. Cell Res. 132, 89-98. Williamson, D.H. (1970) in Control of Organelle Development (Miller, P.L. ed.) Cambrize University Press, Cambridge, pp. 247-276. Grimes, G.W., Mahler, H.R. and Perlman, P.S. (1974) J. Cell Biol. 61, 565-574.

6.

7. 8. 9.

10. 11. 12.

Cottrell, S.F. and Avers, C.J. (1971) in Autonomy and Biogenesis of Mitochondria and Chloroplasts (BoardmanTN.K., Linnane, A.W. and Smillie, R.M. eds.) North-Holland, Amsterdam, pp. 481-491. Williamson, D.H. and Scopes, A.W. (1962) Nature 193, 256-257. Rubin, B.Y. and Blamire, J. (1977) Molec. gen. Genet. 156, 41-47. Cottrell, S.F. and Avers, C.J. (1970) Biochem. Biophys. Res. Commun. 38, 973-980. Casey, J., Hsu, H.J., Rabinowitz, M., Getz, G.S. and Fukuhara, H. (1974) J. Mol. Biol. 88, 717-733. Marmur, J. (1961) J. Mol. Biol. 3, 208-218. Cottrell, S.F., Rabinowitz, M. and Getz, G.S. (1973) Biochemistry 12,

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17.

McCarron, R.J., Cabera, C.V., Esteban, M., McAllister, W.T. and Hdowezak, J.A. (1978) Virology 86, 88-101. Caplen, H.S. and Blamire, J. (1980) Cytobios 29, 115-128. Blanc, H. and Dujon, B. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3942-3946. Goursot, R., de Zamaroczy, M., Baldacci, G. and Bernardi, G. (1980) Current Genet. 1, 173-176. Cottrell, S.F., Rabinowitz, M. and Getz, G.S. (1975) J. Biol. Chem.

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