BIOCHEMICAL

Vol. 69, No. 2, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A DNA LIGASE FROM MITOCHONDRIA OF RAT LIVER Cora J. Levin

and Steven

B. Zimmerman

Laboratory of Molecular Biology National Institute of Arthritis, Metabolism and Digestive Diseases National Institutes of Health Bethesda, Maryland 20014

Received

January

20,1976

SUMMARY: An ATP-dependent DNA ligase has been demonstrated in extracts of rat liver mitochondria. The activity may be released from the mitochondria by treatment with hypotonic solutions or a detergent, indicating an intramitochondrial localization. The properties of the partially purified enzyme are similar to those of the nuclear DNA ligase from rat liver. Several

percent

mitochondria.

This

implying

rat

in the manner

from

rat

cytoplasmic MATERIALS

liver

found

liver.

This

expected

for

of the partially

purified

often

activity

is

for

released

some

contained circular

form

and other

to those

(l), in

in mitochondria

by osmotic

of those

within

one step

localization.

similar

from

is

at least

DNA ligase

an intramitochondrial

differ

cells

in a closed

activity

an ATP-dependent

its

isotreatments

The properties

of the nuclear reported

for

DNA ligase a "soluble"

(4).

AND METHODS:

DNA ligase: DNA ligase and protein DNA ligase Fraction IV was isolated before (3). Mitochondria: steps were

DNA is

enzyme are but

(2,3,4)

ligase

DNA of eukaryotic

of a DNA ligase-like

We have

from

total

mitochondrial

the action

replication. lated

of the

Mitochondria at O-5".

were

were from isolated

assayed as previously the nuclear fraction from

rat

liver

described (3). of rat liver as

by two procedures.

All

Differential centrifugation (5): Liver homogenates were prepared with a Sorvall Omnimixer as before (3) extent a medium (Medium H)_ comvosed of 0.25 M _ sucrose--l mM EDTA (pH 7) (6) was ' used - at 5 ml per g of liver. Immediately before homogenization, 1 volume of 0.07 M phenylmethylsufonyl fluoride (Calbiochem) freshly dissolved in 95% ethanol was added per 35 volumes of Medium H. The homogenate was centrifuged 3 times for 10 min at 600 x g; each time about 314 of the supernatant fluid was decanted taking care not to dislodge loosely packed pellets. The final supernatant fluid was centrifuged for 10 min at 6,000 x g and the pellet resuspended in 100 ml of Medium H. This high speed centrifugation was repeated three times more and finally the pellet was weighed, frozen with Dry Ice, and stored at -70".

Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

514

Vol. 69, No. 2, 1976

BIOCHEMICAL

AND EIOPHYSICAL

RESEARCH COMMUNICATIONS

Step-wise gradient centrifugation: Mitochondria were prepared by differentia centrifugation as above except the final (unfrozen) high speed pellet from 6 rats was resuspended in 9 ml of Medium H and 3 ml aliquots layered on top of stepwise sucrose gradients composed of 12 ml of 1.0 M sucrose-5 mM EDTA-10 mM Tris Cl (pH 7.6) over 12 ml of 1.5 M sucrose-5 mM EDTA-10 mM Tris Cl (pH 7.6) (8). After centrifugation for 70 min at 20,000 rpm (Spinco, SW 25.1) there was a dense mitocondrial layer at the interface of the 1.0 M and 1.5 M sucrose solutions, and much smaller amounts of material as a pellet and at the interface between the 0.25 M and 1 M layers. The mitochondrial layer was removed with a pipet, centrifuged for 10 min at 6,000 x g, resuspended in 30 ml of Medium H and recentrifuged at 6,000 pellet was stored frozen, as above. x g- The final Purification

of the mitochondrial

The extraction and Simpson

Meyer

DNA ligase:

and G-25 filtration (6). All steps were

are directly at O-5".

based

upon the procedure

of

Extraction: A partially thawed pellet of mitochondria was ground for 2 min in a mortar (prechilled to -12') with 2 parts by weight of alumina (A-301, Alcoa) per part of wet weight of pellet. Extraction buffer (0.05 M Tris Cl (pH 8.0)5 mM S-mercaptoethanol-1 mM EDTA-0.05 M Mg acetate-l M NaCl) was added (2 ml/g mitochondrial pellet) and grinding continued for 20 sec. The mixture was centrifuged 30 min at 17,000 x g. The pellet was reextracted by briefly stirring with l/2 the original volume of extraction buffer, recentrifuged, and the supernatant fluids combined (Fraction I, specific activity = 0.3 units/mg, recovery taken as 100%). Sephadex filtration: Fraction I (27 ml) was desalted by passing through a Sephadex G-25 (Pharmacia) column (3.8 cm2 x 40 cm, equilibrated and eluted with 25 mM Tris Cl (pH 8.0)-5 mM B-mercaptoethanol-1 mM EDTA). The excluded volume was pooled (Fraction II, specific act. = 0.3, recovery = 90%). Fraction II was frozen in Dry Ice and stored at -70". Phosphocellulose chromatography: Fraction II (25 ml) was loaded onto a phosphocellulose column (0.9 cm2 x 15 cm, equilibrated with 0.01 M K phosphate (pH 6.8)-3 mM B-mercaptoethanol) and eluted with a linear gradient (0 to 0.6 M KC1 in the equilibration medium). The only ligase activity detected appeared as a sharp peak at about 0.37 M KCl. Peak fractions were pooled (Fraction III, = 76%). Generally, l/10 volume of bovine plasma albumin sP* act. = 16, recovery (10 mg/ml) was added and the Fraction III quick-frozen and stored at -70'; this material was dialyzed (2 hrs., vs. 10 mM Tris Cl (pH 8.0)-10 mM S-mercaptoethanol) before use. Recovery of activity upon frozen storage followed by dialysis was 50-70%. RESULTS AND DISCUSSION: Mitochondrial contain

reproducible

to purify isolated

isolation

and extraction

levels

of ligase

the mitochondria by different

procedures:

irrespective

or to extract

numbers

the

of cycles

specific

activity

in their

after

stepwise

gradient

centrifugation

of mitochondria

gave extracts

with

of the exact

ligase.

the most

515

extracts

procedures

as did

ligase

I).

those

yielded

mitochondria

Alumina activity;

used

mitochondria

centrifugation

(Table stable

of mitochondria

For example,

of differential

the same ligase a further

Extracts

disruption however,

Vol. 69, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I

DNA ligase

activity

mitochondria

Mitochondrial

isolation

in extracts

isolated

prepared

by different

from

procedures

DNA ligase specific activity

procedures

unitslmg Differential

centrifugations

3 low

speed + 4 high

speed

0.18,

4 low

speed + 6 high

speed

0.15

Stepwise

gradient

centrifugation

3 low

speed + 4 high

(after

differential

detergent below)

similar

The latent -______-

ly

in

We have

released

into

solutions

(Fig.

of that

solubilized

a treatment released significantly NaCl

(Fig.

into

Calbiochem)

ligase:

solutions.

found

that

increasing

The release

of ligase

by exposure to release

a non-particulate changed

1, squares

(see

Such extraction

used previousenzymes

of ligase

in Figure

to the

detergent,

components

or after

when

activity

to increasingly

expressed

extraction

516

has been

amounts

membrane-bounded form before

circles).

is

rupture

of mitochondrial

by exposure

of mitochondria

by a subsequent vs.

property

of a number

state

used

shock

Mitochondria This

localization

similarly

and osmotic

of activity.

a non-particulate 1).

often

X-100,

hypotonic an internal

0.28

0.22

of mitochondrial

sufficiently

(9,10,11).

Triton

levels

nature

to demonstrate

are

(0.1%

0.22,

speed

centrifugations)

treatment yielded

placed

protein

osmotic

1 as a percent Triton

(9).

X-100,

The activity

shock was not

of the particles changed

hypotonic

with

the pattern

0.2 M of

Vol. 69, No. 2, 1976

BIOCHEMICAL

o(‘1

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

0.1

0.2 SUCROSE,

0.3

M

Figure 1. Release of DNA ligase and protein into a soluble form by osmotic treatment of mitochondria. Aliquots of mitochondria (3.0 mg total protein) prepared by differential centrifugation (Methods) were pelleted in two series of tubes. The pellets were resuspended in 0.5 ml of a sucrose solution at the concentration indicated. After 10 min at 0", one series of tubes (0,)) was brought to 0.2 M NaCl (by addition of 1 M NaCl) while no addition was made to the other series (El ,I ). After an additional 10 min at O', the tubes were centrifuged 10 min at 10,000 x g and the supernatant fluids assayed for ligase and protein content. One member of each series was similarly extracted with 0.25 M sucrose containing 0.1% Triton X-100; the data are expressed as percentages of the ligase or protein released by this medium (0.13 or 0.17 ligase unit and 1.0 or 1.1 mg protein, respectively, in the two series).

solubilization

of several

membranes

rather

solubilization (Fig.

again

Partial by gradient

where

elutes Properties activity

and of the bulk

with

only

the internal

the protease

inhibitor

eluted

the nuclear

a single

enzyme

--of the mitochondrial (Table

II)

were

the

localization

of the enzyme.

was purified

The total

yield

peak of activity from

(0.36-0.38

ca.

50-fold

of activity

on

was seen except

the homogenization. M) was similar

The to that

M) (3).

ligase: similar

between

of the mitochondria

was omitted

(0.34-0.36

mitochondrial

protein

Ligase

sharp

the activity

onto

The similarity

on phosphocellulose.

was 90-104%;

which

adsorbed

(11).

of the DNA ligase: -__-

chromatography

KC1 concentration

ligase

consistent

they were

internally

activity

purification

such a column

which

localized

of ligase

1) is

in cases

than

enzymes when

to those

517

The requirements of the nuclear

for

mitochondrial

enzyme

(3).

The

Vol. 69, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE II Requirements The complete system dialyzed ligase Fraction

for

mitochondrial

contained III.

DNA ligase

the usual

assay

Components

activity

components

Ligase

Omit

min.

0.029 enzyme

co.003

Omit MgC12

co.003

Omit NaCl

0.004

Omit ATP

co.003

Omit ATP, heated Omit

5 pg of

activity

pmolel20 Complete

plus

add NAD+ (1 mM) or extract

of rat

livera

(0.03

B-mercaptoethanolb add p-Chloro-

mercuribenzoate [32P]DNA

Heated

enzyme

(10 min, (5 min,

~0.003 0.009

Chnit B-mercaptoethanolb,

Heated

ml)

(10 PM)

co.003

100")

co.003

65")

co.003

CompleteC

0.037

Add 1 pg of pancreatic

DNase

co.003

Add 1 ug of pancreatic

RNase

0.041

Add 1 pg of Pronase

0.035

a Extract is supernatant fluid after heating (10 min. 100") a homogenate of rat liver (1 g per 3 ml H20) and centrifuging 10 min at 8,000 x g. An increased blank (0.01 pmole) due to the heated extract has been subtracted. b b-Mercaptoethanol

omitted

from

' After initial 20 min. incubation enzymes indicated and incubate 20 min.

both with 37".

518

dialysis ligase,

and assay heat

media.

5 min.

100";

add

Vol. 69, No. 2, 1976

BIOCHEMICAL

> I= 2

N&I,

1

AND BIOPHYSICAL

M

2

MgCI,,

3

mitochondrial

optimum

ligases

of NaC1, MgC12,

responded

ATP or DNA (Fig.

(pH 8.0 > pH 7.1 or pH 9.0).

was estimated

for

ref.

3, with

that

previously

10

DNA.

pglml

Effects of NaCl, MgC12, ATP or DNA concentrations and nuclear DNA ligases. Mitochondrial ligase,

and nuclear

centration

mM

5

ATP, mM

Figure 2. of mitochondrial ligase, 0.

RESEARCH COMMUNICATIONS

the mitochondrial

5 ml of Fraction estimated

for

similarly

2).

The enzymes

Finally, ligase

II,

very

83% recovery

the nuclear

to changes

a molecular (by gel

in

weight

filtration

rate

con-

had a similar

pH

of 10 x lo4

as described

of activity)

enzyme

upon the 0; nuclear

which

is

the

in same as

(3).

Conclusions: Previous extracts aspects fairly

yielded

only

stable

and Simpson

levels

procedure

There

activity.

ligase

activity

unstable

activity

seem to be responsible

First, Secondly

which

yield

the alumina a protease

in mitochondrial (tl,2 for

reproducible extraction inhibitor

< 1 day).

obtaining

Two

active

and

chromatographic technique

was present

of Meyer during

the

homogenization. are

two types

extracts is

of very

extracts

(6) was used.

cell

chondrial

low

mitochondrial

of ligase

initial

by us to demonstrate

of the present

profiles

activity

attempts

insensitive

of evidence

originates to the

within exact

that

the ligase

activity

the mitochondria. procedure

519

used

to

measured

in mito-

First,

the level

of

purify

the mitochondria.

Vol. 69, No. 2, 1976

Secondly, media

BIOCHEMICAL

the activity

under

is

conditions

known

The mitochondrial all

enzyme

contrast, reported

the

functions "soluble"

is

species

chromatographic isolated

behavior from

rupture

We therefore in both fraction

(4). calf

(4,12)

molecular In addition,

thymus

that

tissue

organelles. the nuclear

from

from

this

distinguishable weight multiple

to hypotonic

ligase

in

the same or a closely

and mitochondria

of the cytoplasm

by greater

these from

suggest

nuclei

RESEARCH COMMUNICATIONS

the mitochondria

indistinguishable

to have one or more ligases

mitochondrial

have been

tested.

by exposing

to osmotically

ligase

the properties

related

released

AND BIOPHYSICAL

rat tissue

from

and somewhat species

liver.

In

has been the nuclear-

different of nuclear

ligase

(13).

REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Borst, P., and Flavell, R. A. (1972) Mitochondria: Biogenesis and Bioenergetics, pp. 1-21, North-Holland, Amsterdam. Tsukada, K., and Ichimura, M. (1971) Biochem. Biophys. Res. Commun., 42, 1156-1161. Zimmerman, S. B., and Levin, C. J. (1975) J. Biol. Chem., 250, 149-155. Teraoka, H., Shimoyachi, M., and Tsukada, K. (1975) FEBS Letters, 54, 217-220. Schneider, W. C. (1948) J. Biol. Chem., 176, 259-266. Meyer, R. and Simpson, M. V. (1968) Proc. Nat. Acad. Sci. U.S.A., 61, 130-137. Fahrney, D. E. and Gold, A. M. (1963) J. Am. Chem. Sot., 85, 997-1000. Bogenhagen, D. and Clayton, D. A. (1974) J. Biol. Chem., 249, 7991-7995. Bendall, D. S. and de Duve, C. (1960) Biochem. J., 74, 444-450. Baudhuin, P., Hertoghe-Lefevre, E., and De Duve, C. (1969) Biochem. Biophys. Res. Commun., 35, 548-555. Baudhuin, P., Peeters-Joris, C., and Bartholeyns, J. (1975) Eur. J. Biochem., 57, 213-220. Austin, G. E. (1975) Fed. Proceedings, 34, 701. SijderhBll, S. and Lindahl, T. (1975) J. Biol. Chem., 250, 8438-8444.

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