Multiple DNA polymerase activity solubilized from higher plant chromatin

BIOCHEMICAL

Vol. 54, No. 1,1973

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

MULTIPLEDNAPOLYMERASE ACTIVITY SOLUBILIZEDFROM HIGHERPI&T CHROMATIN Valgene L. Dunhamand Joe H. Cherry Horticulture

Department, Purdue University

West Lafayette, Received

July

20,

Indiana

47907

1973

DNApolymerase preparations solubilized from chromatin of unwashedand washed sugar beet storage tissue exhibit multiple activity peaks when separated by DEAE-Sephadexchromatograp$y. Activity peaks isolated from unwashed and washed tissue required Mg all four deoxynucleoside triphosphates and added DNA. Activity from peaks is:lated from washed tissue was completely sensitive to pancreatic DNaseand insensitive to RNase. Enzyme activity was increased in the tissue during the washing period. In addition, there was a drastic change in template specificity from single-stranded primer in unwashed tissue to double-stranded DNAin washed tissue. Recent investigations tissues and in different multiple lular

of DNApolymerase activity

in whole eukaryotfc

locations have indicated the presence of 1-4 DNApolymerases having distinct properties . In addition, the cel-

concentration

cellular

of certain of these

of normal or induced growth 5-7 .

enzymes change during specific

In an attempt to elucidate

nucleic acid synthesis in higher plants,

phosphate buffer

increase in nucleic acid synthesis g,9 . and properties

of multiple

the controls of

we have chosen sugar beet storage

tissue as an experimental system. Following slicing for extended time periods in sterile

stages

To investigate

DNApolymerases in plants,

of the tissue, washing results in a dramatic the possible presence the isolation

of chroma-

tin from unwashedand washed sugar beet tissue was selected as a starting point and a source of the enzyme(s). In this communication we report the separation of four peaks of DNApolymerase activity

by DEAE-Sephadexchromatography of enzyme preparations from

both unwashedand washed tissue. at the samesalt concentration

Two of the peaks from washed tissue eluted as two of the peaks from unwashedtissue.

Furthermore, three of the four peaks from washed tissue exhibited

Copyright 0 1973 bv Acudemic Press, Inc. AN rights of reproduction in any form reserved.

403

a marked

Vol. 54, No. 1, 1973

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

preference for double-stranded template, whereas a preference for singlestranded DNAwas observed with activities Materials

from unwashedtissue,

and Methods

Sliced sugar beet roots were prepared and washed in sterile buffer,

phosphate

10

pH 6.5, as described previously.

Chromatin was isolated by a modification

of a procedure described by

Huang and Bonner11 from 9OOglots of unwashedor 20-hour-washed tissue. polymerase activity

was solubilized

through Sephadex G-50 as previously

from chromatin and desalted by passage 12 described for RNA polymerase. Sugar

beet DNAwas isolated by a modification EDTA-SSCfractionation,

DNA

of a procedure"

which included SDS-

pronase and ribonuclease treatment and hydroxylapatite

column chromatography. The soluble DNApolymerase assay contained in a total

volume of 0.4 ml:

40 knoles tris-HCl,

pH 8.0; 2 umoles MgC12; 2 umoles dithiothreitol; 0.1 IJmole 3 each of dATP, dCTP, dGTP; 0.004 pmole TTP, 0.0006 pmole of H-TTP (specific radioactivity

18.8 mc/pmole); 20 ug DNAand 0.05 ml enzyme (corresponding to

lo-15 ug protein).

The reaction

was initiated

with the addition

of enzyme,

run for 30 minutes at 37' and stopped with the addition of 3 ml of 40 mMsodium pyrophosphate in 10%TCA. The assay mixtures were filtered

through glass fil-

ters (WhatmanGP/A) which were then washed repeatedly with 5% TCA, dried, the radioactivity

determined by liquid 14 was by the Lowry method,

scintillation.

and

Protein determination

Results Extract.

Following desalting of the enzyme preparation

G-50 column (2.5 x 7.5 cm), the extract polymerase activity a2+,

was partially

through a Sephadex

characterized.

DNA

isolated from unwashedand washed tissue was dependent on

added DNAand all

four deoxynucleoside triphosphates

was linear through 30 minutes and proportional DUE-Sephadex chromatography.

Extracts

404

(Table 1).

Activity

to added protein up to 50 pg. (from unwashedand washed tissue)

BIOCHEMICAL

Vol. 54, No. 1, 1973

Table

1.

Properties extract

AND BtOPHYSICAL RESEARCH COMMUNICATIONS

of DNA polymerase solubilized

activity

from chromatin.

Unwashed

tissue

Washed tissue

relative Complete* +g2+

+ 12 mM EDTA

dCTP

-dATP,

dCTP, dGTP

100

<1


DNA + native

*Complete:

C. T. DNA

unwashed

30 minutes

washed

were

separately

equilibrated of 0.032 of (NH4)2 were

M (NH4)2

observed

sues appeared III

resulted

Activity

peaks

tissue.

Activity

calf

to columns

to 0.08

15

32

40

TMp incorporated/mg

in a tremendous

eluted

A-25

with

protein/

intirease were

by 50% after

405

were

washed

Four

major

Peaks I and II

at the peak,

(1 x 20 cm) previously

same salt appeared

were

gradient activity

of both

to be absent

solubilized

stable

than

15 ml

peaks tis-

concentration.

Peak in unwashed

from both

in DNA polymerase less

with

an 80 ml linear

1).

tissues.

of protein

tissue

protein/

TMp incorporated/mg

The columns

activity

amounts

was reduced

2

M) in TGMED (Figure

a major

from unwashed

1

pmoles

, pH 7.9.

the columns

from

2

of DEAE-Sephadex

to elute

similar

3

thymus DNA as template).

and washed

Although

washing

= 75.2

SO4 in TGMED and then

tissue,

6

thymus DNA as template).

from unwashed

from washed

tissue.

tissue

TGMED buffer

SO4 (0.032

6

= 25 pmoles

calf

(activated

adsorbed with

tissue

(denatured

30 minutes

18


-DNA -assay

activity

100

-dATP -dATP,

in

activity those

one week in liquid

tissues,

(Figure

1).

from washed nitrogen

for

peaks

Vol. 54, No. 1, 1.973

BIOCHEMICAL

UNWAMEO

0.4

. 0

t

0.4

AND BIOPHYSLCAL RESEARCH COMMUNICATIONS

TISSUE

A2e.o DNA Polymarare

WASHED

TISSUE

IO

20

Activity

0.3

0

Figure

1.

isolated

0

DEAF-Sephadex chromatography from chromatin of unwashed tissue profile: Peak I = III = tubes 36,3? and Peak Peak I = tubes 14-16, Peak and Peak IV = tubes 38-40.

from unwashed

washed

tissue

thawing.

Other

with

two salt

step

fractions

observed

attempts

only

the

10% after

at purification

using

resolved

four

activity

peaks

activity

repeated

DEAF-Sephadex with

the

freezing

in batch

linear

gradients

at the salt

from and

columns on the salt

concentrations

1.

of DNA polymerase from both

deoxynucleoside

of peak I,

two weeks with

have

characterized

60

exception

by chromatography

activity

50

of DNA polymerase activity solubilized and washed sugar beet tissue. Unwashed tubes 14-16, Peak II = tubes 23-25, Peak IV = tubes 40-42. Washed tissue profile: II = tubes 24-26, peak III = tubes 31-34

followed

peaks

through

With

40 NO

steps

activity

linear

tissue.

was reduced

in Figure

Properties

four

30 FRACTION

tissues (Table

were 2).

triphosphates,

45 minutes

activities.

Peak tubes

separately

Activity Mg 2+

pooled

of the four

major

and the DNA polymerase

in each peak was dependent , added

and proportional

with

406

DNA and enzyme. respect

Activity

on all was

to enzyme concentration

BIOCHEMICAL

Vol. 54, No. 1,1973

Table

2.

Properties

AND BIOPHYSICAL

of DNA polymerase

DEAR-Sephadex

RESEARCH COMMUNICATIONS

activity

peaks

following

chromatography.

--^~-----.--_

-Unwashed -----

tissue

Peak I Complete

II

III

IV

15.5*

38.9

11.3

20.3

-m 2+ + 12 mM EDTA

0.5

0.9

1.0

0.2

-dNTP

1.1

0.8

0.0

1.5

-DNA

0.5

0.3

0.2

0.2

-enzyme

0.2

0.1

0.0

0.1

I Complete

Washed tissue --

II

72.6

1936.5

III

IV

1054.2

668.1

-pfg2+ + 12 mM EDTA

0.0

0.0

0.0

6.0

-dNTP

3.0

4.1

1.0

0.6

-DNA

0.0

1.0

1.6

0.8

-enzyme

0.2

0.0

0.1

0.7

--;kActivity

expressed

saturating peak

as pmoles/mg

levels

(see Table

of template

Activity

The products sensitive Activity

Further

due to the high

in all

four

to pancreatic

activity

in peaks

activity

employing for

each

employing

80% of that

was employed

of the activity isolated

sensitive each peak

DNase (20 ug/ml)

was approximately (dA-T)

the most

characterization

peaks was completely

of the reaction

DNA when poly

yielding

30 minutes

3).

(up to 40 cLg in assay). was possible

of protein'in

from washed to actinomycin

separately

and insensitive observed

as template.

407

of each peak

with

were

tissue. D (40 hg/ml).

completely

to RNase (20 us/ml). activated

No activity

calf

thymus

was observed

when

Vol. 54, No. 1, 1973

poly

BIOCHEMICAL

(rA-dT)

was employed

Template

specificity,

separately

studied

requirements procedure.

as the primer/template The four

to determine

and (2)

(1)

whether

this

3.

Template

Specificity

NATIVE C.T.

the peaks

have

was altered

3, there

is

DNA

DNA

12.8*

an extensive

template the washing

shift

activity

peaks. were

different

in overall

peaks.

PEAK

II (100)

tissues

during

of DNA polymerase

J.

C.T.

from both

DEAE-SEPHADEX

TEMPLATE

any of the activity

peaks

specificity

UNWASHED TISSUE

ACTIVATED

for

activity whether

As can be seen in Table

Table

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

III

8.1

(100)

11.3

IV (100)

0.4

( 100)

(

0.0

(

0)

13.0

(161)

0.0

0)

2.6

( 628)

(

0)

38.9

(482)

7.2 ( 63)

20.3

(4842)

DENATURED C.T.

DNA

0.0

DENATURED S.B.

DNA

15.5

(120)

4.5

( 56)

0.0

(

0)

0.0

(

0)

10.0

( 78)

2.0

( 25)

1.3

( 11)

0.0

(

0)

(

0.0

(

1.3

( 10)

0.0

(

0)

NATIVB S.B.

DNA

0.0

YEASTRNA

0)

0)

WASHBEDTISSUE --ACTIVATED NATIVE C.T.

C.T.

DNA

DNA

DENATURED S.B.

DNA

8.4

(

S.B.

31.9 0.0

of protein

for

activity

based

of control = Sugar

( 13)

119.5

( 11)

107.4

(

6)

37.3

(

22.2

(

1)

11.6

134.7

(

7)

0.0

(

0)

(309) ( 10)

*Pmoles/mg

258.3

72.6 2.4

YEAST RNA

(100)

1936.5

DNA

DNA

1054.2

(100)

DENATURED C.T.

NATIVE S.B.

(100)

23.5

4)

(136) (

0)

Numbers

30 minutes. on employment

beet.

408

( 100)

18.5

(

3)

4)

7.1

(

1)

(

1)

31.5

(

5)

35.3

(

3)

23.7

(

4)

0.0

(

0)

1.5

(

1)

in parenthesis

of activated

668.1

calf

represent thymus

percentage

DNA as template.

BIOCHEMICAL

Vol. 54, No. 1,1973

template specificity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

In unwashedtissue there is

during the washing period.

a preference for single-stranded

DNA, especially

noticeable

in peaks II and IV.

In washed tissue, however, double-stranded template is preferred, to the strong preference for activated III

calf thymus DNAexhibited

primarily

due

by peaks II,

and IV. Discussion The slicing

and washing of sugar beet storage tissue results

in increased

DNAsynthesis as evidenced by an increase in DNApolymerase activity. time we do not know if protein in polymerase activity. could result

If

removal of an inhibitor profiles

synthesis is necessary for the observed increase

enzyme activation

from alteration

of existing

is involved,

during the washing period.

resulted from -de novo synthesis,

enzyme activation

may be the sameproteins.

or both.

Peak III

The interesting

could be a result of of an al-

protein.

change in template specificity

following

period may also have resulted from protein alteration DNaseactivity

properties

of washed tissue, however,

This does not, however, preclude the possibility

of an existing

may have

Peaks I and II from

and have similar

in unwashedtissue and its activity

de novo synthesis. -teration

Based on the DEAGSephadex

the increased activity

both tissues elute at the samesalt concentrations

is not detectable

increased activity

polymerase molecules or by the simple

and the various peak characteristics,

and, therefore,

At this

the washing

or an induction

and its association with the polymerase activities.

of The pre-

ference for double-stranded template in washed tissue does not agree with DNA polymerase activity unfractionated

from corn

16

enzymes were found to prefer denatured templates.

is possible that the high activity activated

and from various algae 17,18 in which single

of sugar beet polymerase observed with

calf thymus DNAwas primarily

19 groups , high activity

Al though it

a result

of the exposure of 3' hydroxyl

was also observed when native

sugar beet DNAwas employed as template.

409

calf thymus or native

Vol. 54, No. 1, 1973

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The product of the DNApolymerase reaction as isolated specified

by the base composition of the native primer.

template, however, an increased incorporation studies further

purification

of the activity

sugar beet tissue and further indicate

Employing denatured

of dAMPwas observed.

In our

peaks from unwashedand washed

characterization

of the reaction products should

whether or not the change in template preference results in products

of altered base composition, if the activity

In addition,

studies are underway to determine

peaks are all unique enzymes, if

washing and, ultimately, lication

from corn 16 was

the function

enzyme alterations

occur during

of each of the enzymes in repair or rep-

of the plant genome. Acknowledgements

Wewish to thank Mrs. Jane Thurman for her skilled

technical

assistance.

This work was supported by a grant ROl ES 00551-03 from the National Institute of Health.

This report is journal paper 5202 of the Purdue Agriculture

Exper-

iment Station.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

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