Vol. 66, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

END GROUP LABELLING OF RNA AND DOUBLE STRANDED DNA BY PHOSPHATE EXCHANGE CATALYZED BY BACTERIOPHAGE T4 INDUCED POLYNDCLEOTIDE KINASE George

Chaconas,

Division

Received

Johan

H. van de Sande and Robert

B. Church

of Medical Biochemistry, Faculty of Medicine and Department of Biology The University of Calgary Calgary, Alberta, Canada T2N lN4

August

7,X975

SUMMARY: End group labelling of sheared double-stranded DNA, and tRNA has been effected without prior dephosphorylation, utilizing the reversal of TI+ polynucleotide kinase activity. Incubation of DNA with polynucleotide kinase in the presence and absence of a phosphate acceptor (ADP) allowed the determination of the relative ratio of 5' hydroxyl and 5' phosphoryl terminii in the polynucleotide. This method of analysis has demonstrated a high preference in the formation of 5' vs 3' phosphomonoesters during high pressure shearing of double stranded DNA.

Tg and T4 induced y phosphate (1,

2).

tural

analysis

enzymology

with

(11,

in which

suggested

that

acids

the

lo),

be quantitatively

lysed

phosphate

to end group

(no prior

the end group

that

of the

in the struc4),

fingerprinting

the study

of DNA

a 5’

hydroxyl

the enzyme requkes require

labelling

with

as substrates,

treatment polynucleotide

van de Sande et az.

of polynucleotide

as an ADP dependent

(16)

(3,

as in

and oligonucleotides

labelled

exchange

as well

RNA's

reversibility

transfer

extensively

analysis

Since

eliminating

has been shown

for

(9,

the enzyme could

thereby

could

extended

in sequence

the

of DNA, RNA and oligonucleo-

15).

prior

the enzyme can act

It

terminus

deoxyoligonucleotides

demonstrated

nucleic

lation.

13, 14,

phosphatase

Employing have

finding

12,

catalyze

enzyme has been used

acids,

2) most DNA's,

alkaline

(16)

been

induced

of nucleic

(1,

kinase.

ryl

The%

6, 7, 8) and gene synthesis

terminus

kinases

from ATP to the 5' hydroxyl

tides

(5,

polynucleotide

kinase

activity,

phosphomonoesterase.

be used to directly the necessity

for

label prior

This 5' phospho-

dephosphory-

a deoxy-5'-phosphoro-nonacosanucleotide

by means ofTqpolynucleotide dephosphorylation). labelling

of double

kinase These

stranded

findings

catahave

DNA and tRNA.

Vol. 66, No. 3, 1975

BIOCHEMICAL

Moreover,

the described

distribution

of 5'

shearing

of high

method

AND BIOPHYSICAL

permitted

and 3' phosphomonoesters

molecular

weight

double

MATERIALS

the direct formed stranded

RESEARCH COMMUNICATIONS

analysis during

of the high

relative

pressure

DNA.

AND METHODS

TL+ induced polynucleotide kinase was purified as Enzymes. Bacteriophage described previously (17). The enzyme preparation showed a single band of on analysis by SDS polyacrylamide gel molecular weight - 35,000 daltons electrophoresis and did not contain any detectable DNAse or RNAse activity. Bacterial alkaline phosphatase (BAPC) obtained from Worthington Biochemicals was further purified by DEAE-cellulose chromatography as described by Weiss et ~2. (11).

PoZynucZeotides.

DNA was extracted from E. cozi K12 by a modification of the Marmur procedure (18) and calf thymus DNA was purchased from Worthington Biochemicals. E. coli tRNAva1 (Boehringer) was a gift from Dr. G. H. Dixon and crude yeast tRNA (General Biochemicals) was a gift from Dr. I. Forrester. DNA sheared by passage through a needle valve at 15,000 p.s.i. displayed the same hyperchromicity as unsheared DNA. [Y-~~P) ATP was prepared by the method of Schendel and Wells (19) or by a modification of the procedure of The ATP was found to contain 0.4% 32Pi, as analyzed Glynn and Chappell (20). by descending chromatography on DE-81 strips in 0.35 M ammonium for-mate. The specific activity of the [Y-~~P] ATP (240 Ci/mmole) was determined upon preparation by two methods developed in this laboratory (21). A DNA marker system consisting of oligomers of d-32pT(pT)9 was kindly provided by B. W. Kalisch (22). DNA was dephosphorylated by incubation of 35-50 nmoles of DNA nucleotide in 25 mM Tris-HCl, pH8.3 at 65'C for 45 minutes. Alkaline phosphatase (0.01 units) was added at 0, 15 and 30 minutes. Control DNA was incubated under the same conditions in the absence of the phosphomonoesterase. The alkaline phosphatase was inactivated by treatment with NaOH (23) or Nitrilotriacetic Acid (24), or the activity was inhibited by the addition of potassium phosphate to a final concentration of 4mM in the kinase reaction mixture All three methods were equally effective in removing the phosphatase (11). activity, in the polynucleotide kinas shown by the absence of 32Pi release ase reactions.

Enzymatic Reactions.

Phosphorylation reactions were carried out in a volume of 0.1 ml containing 50 mM Tris-HCl, pH 7.6, 2-30 nMoles of DNA or RNA nucleotide, 10 mM dithiothreitol, 5 mM MgC12, 7.5 PM [Y-~~P] ATP and 17.5 units of polynucleotide kinase. The exchange reactions also contained 7.5 PM ADP as a phosphate acceptor. The kinetics of the reactions were followed by the removal of 5~1 aliquots and monitoring TCA precipitable counts on Whatman 3MM filters. The reactions were stopped by addition of EDTA to 25 mM followed by boiling the reaction mixture for 2 min. or by extracting the reaction mixture with chloroform:octanol (1O:l).

Isolation and tiaracterization of 32P LabeLled DNAand RNA. Phosphorylated nucleic acids were separated from unreacted [Y-~~P] ATP by chromatography of the terminated kinase reaction mixutes on Sephadex G-200 or G-75 columns prepared in disposable 10 ml pipettes. The columns were equilibrated and eluted with 0.05 M triethylammonium bicarbonate. Fractions (0.1 ml) were collected in small glass vials and the radioactivity was determined by measuring Cerenkov radiation in a Beckman liquid scintillation counter. The fractions were concentrated by evaporation in vacua and subsequently used for analysis on sucrose gradients and polyacrylamide gels.

963

Vol. 66, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Alkaline sucrose gradients (5-20%) were prepared in 0.l.M NaOH, 0.9M NaCl and 1 mM EDTA. DNA phosphorylated using the polynucleotide kinase reaction and purified as above was mixed with identical unphosphorylated DNA as an O.D. marker and layered on the 12 ml gradients. The material was centrifuged in a Beckman SW 41 rotor at 35,000 rpm for 16 hours at 4OC. The gradients were punctured and fractions (0.3 ml) were collected by gravity flow. Radioactivity was determined by measuring Cerenkov radiation and the optical density at 260 nm was determined using a Beckman Acta III spectrophotometer. out

Polyacrylamide gel electrophoresis of phosphorylated in 10% gels in the presence of 7M urea as previously

tRNA was carried described (25).

RESULTS AND DISCUSSION In agreement phosphate possible

with

the reported

TL, polynucleotide

exchange

with

single-stranded

to obtain

near

quantitative

this

method.

tion

into

tide

kinase

Figure

sheared under

The reaction

1 shows the kinetics

standard

containing

than

the reaction

lation

between

oligonucleotides labelling

double-stranded

ADP.

hydroxyl

and 5’ phosphoryl

the high

molecular

a six

reflects

terminii

weight

DNA.

The great

polynucleotides

similar

for

This esters using

observed

preference

is

of primary calf

versus

thymus

that

initial

of radioactivity

in

polynucleoof ADP.

of labelling

of phosphory-

distribution pressure for

high

by sonic

of 5' shearing

of

the

formation

of

pressure

shearing

is

(26,

27).

irradiation

stability

of the phosphate

Similar

phosphorylation

transfer

was followed

phosphate into

DNA.

This

This

has been minimized

method

the extent

preference

alcohols.

with

extent

high

the direct

exchange

Nevertheless,

during

incorpora-

and absence

relative

due to the greater

ATP catalyzed exchange

phosphate

greater

during

DNA using

incubation

in

DNA fragmented

secondary

has been

results

were

obtained

DNA.

It was found DNA a fast

expected

fold

the

formed

5' phosphoryl-terminated to that

DNA after

The difference

the two reactions

it

of double-stranded

in the presence

ADP displayed

catalyzed

(16),

of radioactive

E.coZi

conditions

lacking

kinase

reaction

the determination

is a maximal

slow where

incorporation

of the number

estimate.

964

by a slow is

ATP can act

by decreasing

(no ADP) of sheared

the

incorporation

probably

due to the

as a phosphate ratio

acceptor

of ATP to substrate.

of 5' hydroxyl

groups

by this

(16).

Vol. 66, No. 3,1975

BIOCHEMICAL

AND BIOPHYSICAL

30

Figure

1.

1 also

the sheared

labelling with

using

the

shows

the

E, coli

the result

leaving

in

phosphorylation

relatively in comparison

kinase large

kinase.

The difference

monoesterase,

with

was slightly

of incomplete

polynucleotide

two step

DNA was treated

procedure

by exchange.

the direct

conventional

the polynucleotide

two step

labelling bably

so

The kinetics of phosphorylation of sheared E. coli DNA. The reacThe tions were carried out as described in Materials and Methods. straight phosphorylation (o-o-o) contained 238 FIM DNA nucleotide which had been heated at 65O/45 min. as a phosphatase control. The exchange reaction (o-o-o) contained 47.6 PM DNA nucleotide also heated at 65'145 min. and the final reaction (x-x-x) contained 47.6 uM DNA nucleotide previously dephosphorylated as described in Materials and Methods. The kinetics were followed by removing 5 pliter aliquots and monitoring TCA precipitable counts on Whatman 3 MM filters. The reactions were started by the addition of 17.5 units of polynucleotide kinase after removal of the zero time aliquots. An additional 5.0 units of enzyme were added at 60 minutes. The data have been normalized to adjust for the differences in the amounts of DNA in the reaction mixtures.

Figure which

60 Ttme (mm)

RESEARCH COMMUNICATIONS

amounts

in the

extent

dephosphorylation

using

is

required

of enzyme required

965

prior

to

of phosphorylation

that

observed

of labelling

which also

double-stranded

of enzyme were

in

with

direct

was pro-

of the DNA by the phospho-

terminii It

procedure

phosphatase

The extent than

reaction.

to the amount

alkaline

lower

some 5' phosphoryl

reactions

labelling

would

noteworthy

not that

be labelled in all

DNA as a substrate, to obtain

to obtain

plateau quantitative

levels label-

Vol. 66, No. 3, 1975

BIOCHEMICAL

ling

oligonucleotides.

of single-stranded Labelled

This

DNA was isolated

figure

sheared

also

calf

phosphate

exchange

The frame

shift

between

DNA which

effect

labelling

Since

high

which

differ

results

in their the shorter

activity

per

profiles

were

obtained

of ADP as well

as for

prior

shearing sizes,

for

kinase

unlabelled,

2 (the

lack

in

chain

in the production

polynucleotide

kinase

than

the larger

DNA which

had been

catalyzed

with

alkaline

of the

of fragments labelling specific

Identical

in the presence treated

DNA.

length.

to a higher

fragments.

DNA phosphorylated

sheared

is a result

have a variance

labelled

catalyzed

of correspondence

profiles)

being

2.

of isolated

of DNA results

DNA fragments

mole nucleotide

identical,

and radioactivity which

profile

by polynucleotide

in Figure

of fragments

pressure

in

with

as shown in Figure

gradient

was labelled

observed

density

filtration,

sucrose

and centrifuged

the optical

end group

by gel

shows an alkaline

thymus

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

gradient

or absence phosphatase

to labelling.

0 -

I.5 -

OFRACTION

Figure

2.

NUMBER

Alkaline sucrose gradient profile of sheared calf thymus DNA labelled by polynucleotide kinase catalyzed phosphate exchange and purified on a Sephadex G-200 column (shown in the inset). Identical unphosphorylated, sheared calf thymus DNA was included in the gradient as an 0.1). marker. Details are described in Materials and Methods.

966

Vol. 66, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL

AB

Figure

3.

C

RESEARCH COMMUNICATIONS

D

Autoradiograph of 10% - 7 M urea polyacrylamide gel (20X30 cm) of 32pT(pT)g (slot A) along with a marker system of [32pT(pT)g]n (where n=l, 2, 3, 4.... ) (slot B) and phosphorylated E. coli tRNAva1 (slot C) and yeast tRNA (slot D). The tRNA's were labelled by polynucleotide kinase catalyzed phosphate exchange and purified from unreacted [V-~~P]ATP as described in Materials and Methods. The gel was run at room temperature for 20 hours at 200 mV.

967

Vol. 66, No. 3, 1975

also

BIOCHEMICAL

The polynucleotide

kinase

been used

crude

to label

of phosphorylation increased were

of these

upon

further

purified

from

electrophoresis system denaturing

per

unreacted

showed because

that

both

of their

mole nucleotide

which

were

stronger

can be used

tRNA's

the

to directly

prior

nucleic

acids

necessitates

plateau

levels

that

and may complicate et al.

level

exchange

by running

possible

to minimize

or by using

has resulted

in

specific

hybridization

labelling

the development

whose is

currently

968

be

It

of the

28, unpublished

and easily

investigation.

of ADP

terminii. levels

of

is also

at a higher

use in DNA reassociation under

also

of low

DNA by the methods

of a reliable

should

5' hydroxyl

at O°C.

that

of DNA results

in the absence

by working

(16,

of sheared

It

only

reactions

9.5 while

ratio

activity

shearing

the elimination

reaction is

concentration.

of enzyme to reach

may occur

to label

kinase

reaction

radioactivity

double-stranded

pressure

reported

the reversal

experiments

kinase

quantities

exchange

designed

electro-

autoradiogram

molar

of 5' phosphomonoesters.

polynucleotide

radioactivity

their

manner

RNA fragments

the

on

Under

The use of double-stranded

large

a low ATP to substrate

The end group

from

and high

have recently

pH of the forward

bands

dephosphorylation.

phosphate

by

A DNA marker

by gel

specific

5' phosphoryl-terminated

extremely

3).

expected

to a high

of TI, polynucleotide

experiments (28)

in the

small

be expected

formation low

(Figure

contained

exhibited

of phosphorylation,

in the preferential

and analyzed

The analysis

RNA preparations

label

without

emphasized

markers.

reversal

not be

was run on the same gel. migrated

labelled

could

RNA preparations

filtration gel

has

The kinetics

which

The labelled

by gel

reaction

tRNAva1.

levels

polyacrylamide

labelled

were

exchange

plateau

of d- 32pT(pT)g

than would

acids

of high

M urea

size

nucleic

optimum

[V-~~P]ATP

and therefore

In conclusion,

Okazaki

reached

of enzyme.

of polymers the

phosphate

tHNA and E. cozi

to the oligothymidilate

phoresis which

yeast

addition

conditions

in relation

catalyzed

RNA's

in a 10X-7

consisting

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

pH (the

reverse

is

6.5)

results). described

prepared

above DNA probe

and RNA-DNA

Vol. 66, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL

This Medical Vipond

RESEARCH COMMUNICATIONS

work was supported by the National Research Council The authors are grateful Research Council of Canada. for valuable technical assistance.

and the to Ms. Anne

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

Novogrodsky, A. and Hurwitz, J. (1966) J. BioZ. Chem. 241, 2923. Richardson, C.C. (1965) hoc. Nat. Acad. Sci. U.S. 54, 158. Weiss, B. and Richardson, C.C. (1967) J. Mol. Biol. 23, 405. Jay, E., Bambara, R., Padmanabhan, R. and Wu, R. (1974) Nucleic Acid Res. 1, 331. Szdkely, M. and Sanger, F. (1969) J. MoZ. BioZ. 43, 607. Southern, E.M. (1970) Nature (London) 227, 794. Murray, K. (1973) Biochem. J. 131, 569. Simsek, M., Ziegenmeyer, J., Heckman, J., and RajBhandary, U.L. (1973) Proc. Nat. Aead. Sci. U.S. 70, 1041. Agarwal, K.L., B&hi, H., Caruthers, M.H., Gupta, N., Kleppe, K., Kumar, A ., Ohtsuka, E., RajBhandary, U.L., van de Sande, J.H., Sgaramella, V., Weber, H., Yamada, T. and Khorana, H.G. (1970) Nature 227, 27. Caruthers, M.H., Agarwal, K., Besmer, P., Cashion, P., Fridkin, M., R., Minamoto, K., Rama Moorty, Jay, E., Kumar, A., Loewen, P., Miller, B ., Panet, A., van de Sande, J.H., Sekia, T., Siderova, N., Khorana, H.G. (1973) Ninth International Congress of Biochemistry, Stockholm, Abs. 3010, p. 194. Weiss, B., Live, T.R., and Richardson, C.C. (1968) J. BioZ. Chem. 243, 4530. Gefter, M.L., Becker, A., and Hurwitz, J. (1967) Proc. Nat. Acad. Sci. U.S. 58, 1723. Olivera, B.M. and Lehman, G.R. (1967) Proc. Nat. Aead. Sci. U.S. 57, 1426. Becker, A. and Hurwitz, J. (1967) J. BioZ. Chem. 242, 3685. Weiss, B., Jacquemin-Sablon, A., Live, T.R., Fareed, G.C. and Richardson, C.C. (1968) J. BioZ. &em. 243, 4543. van de Sande, J.H., Kleppe, K. and Khorana, H.G. (1973) Biochemistry 12, 5050. Panet, A., van de Sande, J.H., Loewen, P.C., Khorana, H.G., Raae, A.J. Lillehaug, J.R. and Kleppe, K. (1973) Biochemistry l2, 5045. Marmur, J. (1961) J. Mol. Biol. 3, 208. Schendel, P.F. and Wells, R.D. (1973) J. Biol. Chem. 248, 8319. Glynn, G.M. and Chappell, J.B. (1964) Biochemical J. 90, 147. Chaconas, G., van de Sande, J.H. and Church, R.B. Anal. Biochem., in press. Kalisch, B.W. and van de Sande, J.H. Manuscript in preparation. Jacquemin-Sablon, A. and Richardson, C.C. (1970) J. Mol. BioZ. 47, 477. Simsek, M., Ziegenmeyer, J., Heckman, J. and RajBhandary, U. L. (1973) Proc. Nat. Acad. Sci. U.S. 70, 1041. Maniatis, T., Jeffrey, A., and van de Sande, J.H. Biochemistry, in press. Richards, O.C. and Boyer, P.D. (1965) J. Mol. BioZ. 11, 327. Richardson, C.C. (1966) J. Mol. BioZ. 15, 49. Okazaki, R., Hirose, S., Okazaki, T., Ogawa, T. and Kurosawa, Y. (1975) Biochem. Biophys. Res. Corrrn. 62, 1018.

969