Denaturation of DNA after X-irradiation in vitro

Vpl. 40, No. 4, 1970

BIOCHEMICAL

AND BlOPHYSlCAL

RESEARCH COMMUNlCATlONS

DENATmATIONOF DNA AFTEB X-IFBADIATIOhTVITRO Hanjit

Das Gupta and Sankar Mitra

Department of Microbiology Sose Institute, Calcutta 9, India

ReceivedJune24, 1970 X-irradiation of E. coli DNA caused partial strandseparation as judged by sus?%pmiity of the DNA to nuclease specific for single-stranded DNA, namely an endonuclease from Neurospora crassa and exonuclease I of E. coli. The elution profile of X-irradiated DNA from hydroxyapatite column showed that single-stranded fragments were produced. These fragments had a sedimentation coefficient of 3-4s corresponding to a length of about 250 nucleotides. suMMJmY:

The primary target bacteria

is believed

X-irradiation (b) alteration

of X-ray-induced

to be DNA (1).

and viscosity

(2-S).

trate

Among these electrometric

The measurement of viscosity from strand-breakage

changes in hydrodynamic parameters.

An increase in the ultraviolet

absorbance of DNA as a result

after X-irradiation

tion of bases (2).

(I-5).

spectroscopy,

strand-separation

because both cause similar, denaturation

on DNA of

of DNA after X-irradiation

was studied by ultraviolet

could not distinguish

report

The main effects

and (d) strand-separation

phenomena, the strand-separation titration

damage in

both --in vivo and --in vitro are (a) strand scissions, and elimination of bases, (c) intra- and inter-

strand cross links in vitro

lethal

could be offset

of

by the destruc-

In the present communication, we wish to

the use of enzymatic assay and chromatography to demonsthe formation

of single-strand

stranded DNA as a result

fragments from double-

of X-irradiation. 793

Vol. 40, No. 4, 1970

MAT&3IALS

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNKATION$

AND M.WEODS

32P-labeled modified

MJA was isolated

procedure

low-phosphate

medium

109 ml medium) 1-2 x ,06 molar

of "larmur

was added.

cpm/p.mole.

equivalent

Na-phosphate, a Mullard

method

hour

were

X-ray

further

there

was no after

after

X-irradiation.

upto

per minute.

After

chilled

to 0'

effect

at 80 KVp without FeSOq dosimetry

irradiation, and left

was purified

An endonuclease

the DNA for

showed

for

from Neuromra --I_ crassa (10).

that

E. JYJO~ B accor-

singlemycelia

These nucleases

products

was necessary

an

of D?;A

from

specific

of Linn

about

on hydroxyapatite

DNA to acid-soluble which

operated

properties

(9).

to an extent

to degrade

90 to

of 10%

of

DNA. The incubation :

denatured), MgC12,

as

at room temperature

physical

the

of chromatography

at an amount

detiatured

mC per

!.fm/ml in G.GSM

(8)

stage

native

0.2

Freifelder

I of -E. coli

to the procedure

degraded

follows

in

DNA was purified

according

1-Y:

the

to Lehman

stranded

a

of the D?,YAwas

by Fricke's

processing.

Exonuclease

ding

atmosphere MG 150

generator,

were immediately

cells

a t about

in ordinary

was 3.2 Krads

before

in

of DNA was expressed

irradiated

The dose as estimated

samples

32P (0.3

activity

The concentration

PI; 6.8

any filter.

were grown

carrier-free

The specific

B by a slightly

of phosphorus.

Dfi1A samples

from

i3. col&

The bacteria

(6).

to which

(7)

from

For

exonuclease

20 nyl.moles;

2 kmoles,

conditions I,

for

the

enzymes were

32P DNA (irradiated,

Na-glycinate

8-mercaptoethanol

buffer, 0.3

794

native pI? 9.5,

&mole and the

as or heat-

30 trmoles;

enzyme

in an

Vol. 40, No. 4, 1970

BIOCHEMICAL

amount sufficient

RESEARCH COMMUNICATIONS

to degrade 95% of heat-denatured

volume of 0.3 ml.

D1JAin a total

In the case of Neurospora crassa endonuclease,

mixture was, 32 P-DXA, 20 m!fmoles; Tris-Cl

the reaction 30

AND BIOPHYSICAL

pH 7.5,

yrmoles; MgC12, 3 \tmoles and the enzyme in an amount necessary

to degrade 9O$ of the denatured DNA in a total In both cases, after

incubation

volume of 0.3 ml.

at 37' for 30 min, the reaction

mixtures were chilled,

0.1 ml unlabeled and crude Z. -- coli DNA (2.5 mg[ml) and 0.4 ml 7% HC101,were added. After IO min. at O", the precipitated 10

DNA was removed by centrifugation

min) in the cold and the radioactivity

(10,000 x g,

of an aliquot

of the

supernatant was counted in an end-window GM-counter (DAE, India). Hydroxyapatite

was prepared according to the method of

Miyazawa and Thomas (11).

Hydroxyapatite

columns (3.5 cm x

1.2 cm> were used to separate double-stranded stranded DNA either linear

gradient

by stepwise elution

(total

phosphate, pH 6.8.

1-2

or by elution

with a

volume of 200 ml) of 0.05 to O.&F! Naml of DNA corresponding to 6x10~ to IO5

cpm was loaded on the column. about 95%.

from single-

The recovery of radioactivity

was

The denatured and the native DNA were eluted at about

0.19M and 0.26M Na-phosphate respectively.

Table 1.

Susceptibility of X-irradiated DNA to nucleases specific for single-stranded DNA.

X-ray dose (Kilorads)

9.6

Susceptibility to E. coli exonuclease I (Per cent) 4.7

Susceptibility to Neurosporq crassa endonuclease (Per cent)

5.6

'795

Vol. 40, No. 4, 1970

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNKATIONS

-BZWLTS __----_A&D DWJJSSI@ X-irradiated both

Neurosuora

The extent Since in

endonuclease

of degradation

the

the

This

strand

and double-strand

X-ray

used,

least

in

is

I should

This

clease

not

or DNA partially presumably

because

photoproducts

(Fig.?).

It

presence

that,

to the position

and was found

to be mostly

endonuclease.

Thus its

The correspondence tible

to

fragments

of

the nucleases from

the

there

total

susceptible

and the

dimers

such exonuDNA

and other

in hydroxyapatite

is no radioactivity DNA in the

DNA was eluted

of denatured

amount

that

the

- irradiation,

chromatographed

of total. fraction

hydroxyapatite

column 796

unirradi-

as denatured

The fraction DNA was concentrated

(92%) to Meurosuora

single-stranded the

at

because

fact

of

Unpublished).

DNA was chromatographed.

corresponding

at the dose

to degrade

the

of thymine

of denatured the

3’-

single-

place

of U.V.

while

position

free

single-stranded

as a result

DNA, 4.5% of

DNA when irradiated

of

u.v.-irradiated

and Mitra,

evident

to the

control

in view

DNA was then

is

corresponding ated

to be unable

of the

is clear

base destruction

be expected

(Dasgupta

X-irradiated

Also,

of DNA has taken

denatured

it have

regions

degrade

anywhere degrades

extensive

significant

is pertinent

comment

I could

that

bonds

would

in DITA (I).

I).

were comparable.

(9,10),

causes

to

I (Table

exonuclease

St-ends

X-ray

breaks

denatured

the

regions

because

is unlikely

the

exonulcease DYA ‘ .

likely

it

the

single-stranded

ends,

exonuclease

two enzymes

while

from

susceptible

phosphodiester

regions,

DBA only

of the

and E. _ coli

hydrolyzes

single-stranded

most

to be partially

by these

endonuclease

single-stranded that

DNA was found

nature

was confirmed.

irradiated eluted indicates

DNA suscep-

as single-stranded that

the ,frag-

Vol. 40, No. 4, 1970

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

40:

FRACTION

NUMBER

Gradient elution of %, QJJJ. DNA from hydroxyapatite Fig.1. DRA (A-A); Heat-denatured DNA (0-O); column . Native X-irradiated DBA (9.6 Cads), (0-O).

ments

were

strand

produced

and/or

experiments,

double-strand particularly

irradiated,

beside

DNA was obtained the

position

DNA was also the Neurospora single-stranded existing cular

by simultaneous breakage

which

of double-stranded susceptible

is possible,

were produced breaks,

very

while

in

few single-strand 797

from the

This

DNA. degree that

DNA with

by

the pre-

case of high breaks,

was

in between

to a varying

Thus it

In other

denatured

hydroxyapatite

to degradation

fragments

partially

and single-stranded

endonuclease.

DNA with

from

DNA.

DNA of T7 phage

'fragments,

was eluted

and single-

of irradiated

when homogeneous

single-stranded

single-strand weight

denaturation

mole-

single-

Vol. 40, No. 4, 1970

stranded

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

fragments

single

strands

of the

X-irradiation

centrifugation

with

cient

as compared

tion,

with

9.6

after

single-stranded

2).

about

DNA fragments

Krads,

DNA was reduced

26s as the

to 6.5s

fragments

was about

molecular

weight

the

range

of 7 to 8x10~

nucleotides.

X-irradiation

BOTTOM4

reference

the

3-4s.

of the

8

E.

coli

average

that

daltons,

After

irradia-

of the

formula

to about

20

used here

24

26

native DNA

(121,

was calculated

dose level

16

coeffi-

single-stranded

corresponding

at the

12 FRACTJOM

DNA.

Studier's

fragments

gradient

DPA had a broad

coefficient

of the

Applying latter

produced

sedimentation

T7 phage

sedimentation

while

protruding

by sucrose-density

The native

to the

DI?A with

X-irradiation.

was investigated (Fig.

distribution

as double-stranded

were obtained

The size after

as well

the

to be in 250 increased

32

NUMBER

Fig.2. Sucrose-gradient centrifugation profile of X-irradiated DNA. Fractions corresponding to single-stranded and doublestranded DNA after hydroxyapatite chromatography were separately pooled and concentrated b dialysis against 60% sucrose in 0.05M Na-phosphate, pH 6. B and was subsequently layered on 5-20s linear gradient of sucrose in IM NaC1, 0.0114 Tris-Cl, Centrifugation was carried out in Sti 39 O.OOM EDTA, pH 7.5’. swinging out rotor of Spinco ultracentrifuge, model L at 37,000 rpm for 240 min at lo. Fractions of equal volume were collected from 8 he bottom. The arrow indicates the position of T7 phage DNA (S20,w = 31s). X-irradiated DNA; c----J, singlestranded & a

DNA; (O-O), double-stranded DNA (A-A.) is also shown. 798

DNA.

T!,e unirradiated

yol. 4QNo.4,

BIOCHEMICAL

197!!

the acid-soluble

radioactivity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

only slightly

and hence very small

fragments could not have been produced. The mechanism of X-ray induced denaturation long stretch --et al.

of pol.ynucleotide

chains is not understood.

(2) suggested that X-irradiation

of single-stranded

over such a Scholes

might cause the formation

fragments but they estimated that the number

of hydrogen bond pairs broken per chain break would be of the order of 14 to 16.

Our figure

higher than the above estimate. extent of denaturation

is at least

an order of magnitude

It is not clear whether the

is dependent on the wavelength of X-ray

used. ACKNOWLEDGEMENT The authors wish to thank the Council of Scientific Industrial

Research, India for a research grant.

Bhattacharjee kindly

of Saha Institute

made available

their

and

Dr. S.B.

of Nuclear Physics, Calcutta

X-ray generator.

REFERENCES ::

Freifelder, D., Radiation Res. Suppl. 6, 80 (1966). Scholes, G., Ward, J.F. and Weiss, J.J., J. Mol. Biol., 379

2,

(1960).

Cox, R.A., Overend, W.G., Peacocke, A.R. and Wilson, S., Proc. Roy. SOC. B., 169, 511 (1958). 4. Peacocke, A.R. and Preston, B.N,., Proc. Roy. Sot. B., m, 3.

133 (1960).

5. Collyns, B., Okada, S., Scholes, G., Weiss, J.J. and Wheeler, C.M., Radiation Res., 3, 526 (1965). Marmur, J., J. Mol. Biol., 1, 208 (1961). ;: Thomas, C.A. and Abelson, J., in Proc. Nucleic Acid Res., G.L. Cantoni and D.R. Davies (Editors), Harper and Row, New York, 1966, P. 553. Freifelder, D., Proc. Nat. Acad. Sci. (USA), 2, 128 (1965). ;: Lehman, I.R., in Proc. Nucleic Acid Res., G.L. Cantoni and D.R# Davies (Editors) Harper and Row, New York, 1966, p.203. IO. Linn, S., in Methods in Enzymology, L.. Grossman and K. Moldave (Editors), vol. XII, Academic Press, New York, 1967, p. 247. 11. Miyazawa, Y. and Thomas, C.A., J. Mol. Biol., II, 223 (1965). 12. Studier, F.W., J. Mol. Biol., 11, 373 (1965). 799