Repair of deaminated cytosine residues of DNA: Biological significance of the absence of uracil from DNA

Vol. 83, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Pages 1312-1318

August 29,1978

REPAIR OF DEAMINATED CYTOSINE RESIDUES OF DNA: BIOLOGICAL

SIGNIFICANCE Hiroshi

OF THE ABSENCE OF URACIL FROM DNA

Hayakawa

and

Department of Biology, Kyushu University 33, Received

June

Sekiguchi

Mutsuo

Faculty Fukuoka

of Science, 812, Japan

28,1978

SUMMARY: Uracil-DNA glycosylase of Bacillus subtilis is involved in repair of deaminated cytosine residues of DNA. Survivals of SP02 phage after treatment with bisulfite and weak alkali are considerably higher in wild type strains than in 9 mutants, which are deficient in the enzyme activity, whereas survivals of bisulfite/alkali-treated PBS1 phage in the two types of cells are essentially the same. The spontaneous mutation frequency of a x mutant is three fold higher than is that of a wild type strain. Although structure,

uracil uracil

cells

possess

uracil

into

contain

if

the

cells

host

occuring

in DNA.

and the

deamination

(4,5).

The conversion

reverse

Thus, the

organisms glycosyl

it

GU to

Recently,

of

bond between

glYcosylase

(10) the

that

is

indeed

base act

the

uracil

or through

by heat

even

under (GC)

which for

responsible

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

DNAs which

activity cells

or chemical

having

normally treatment, conditions

to guanine-uracil

is

sometimes to have

and to degrade

of

bases

cells

uracil-DNA

(GU) base

fatal

to

the

a mechanism

site

for

thereby

may be repaired

pathways Here

DNA were glycosylase,

and deoxyribose, other

of

to

pair.

enzymes,

site).

that

transcription

physiological

on uracil-containing

(e.

releasing insertion

evidence

the

of deaminated

1312

in various

the

the base

by an excision

g. direct

repair

found cleaves

we present

0006-291X/78/0834-1312$01.00/0 Copyright All rights

four

to uracil

mutation,

apyrimidinic

apyrimidinic

of

the

revealed

phage

for

labile

Watson-Crick inclusion

both

advantage

guanine-cytosine

original

One of

The resulting to

the

may be advantageous the

that for

in an enzyme

DNA?

occurs

proper studies

permanent

moreover,

their

converted cytosine

enzymes

(6-9).

mechanism base

is of

the

deficient

to be the most

the

Recent

competent

What is in

would cause a transition

organism.

DNA.

It

are

are

DNA (2,3). is known

shown,

thymine

of uracil,

to form

in DNA.

to prevent

has been of

instead

Cytosine

pair

It

in place

uracil-containing

adenine found

mechanisms

DNA (1).

replication

with

usually

elaborate

uracil

thymine,

can pair is not

that

from

repair

of a proper uracilCYtOsine

DNA

BIOCHEMICAL

Vol. 83, No. 4, 1978

residues normal

of DNA.

This

constituent

suggests

AND BIOPHYSICAL

that

the

may be prerequisite

absence

RESEARCH COMMUNICATIONS

of uracil

to organisms

from

DNA as the

to preserve

their

genetic

information. MATERIALS AND METHODS Bacteria and phages: Bacillus subtilis strains used in these experiments were provided by Drs. F. Makino and N. Munakata of National Cancer Center Research Institute. Bacteriophage SPO2cl and PBS1 were obtained from Dr. S. Okubo of Osaka University and Dr. HT Tanooka of National Cancer Center Research Institute, respectively. Media: Broth contained 8 g of Difco nutrient broth, 5 g of yeast extract, 5 mm01 of MgSOb, 20 nmol of MnC12 per liter (pH 7.2). To prepare soft agar and plate, 8 g and 15 g (per liter) of agar were added, respectively. Sedimentation analysis: [3H]Thymine-labeled @X174 DNA was isolated from phage particles by phenol treatment (ll), and treated with an extract of B. subtilis. To prepare the extract, B. subtilis cells were grown in broth at 37'C, harvested during the logarithmic phase, and disrupted in an ultrasonic disintegrator. The DNA samples were layered on 4.7 ml of alkaline sucrose gradients, and centrifuged at 36,000 r.p.m. for 5 hr. Other procedures were as described (12). Host cell reactivation: Phages treated with various agents were diluted with Tris dilution fluid (PH 9.0). which contains 5.4 e. of NaCl. 3 z of KCl. 1.1 & of NHkCl, 12.1 g of iris, C;l mm01 of CaC12, 1 mm01 of MgSO+,-1 nmol ' of FeC13, 20 pmol MnClz in 1 liter of water. Analiquot(O.l ml) of phage suspension was plated with 0.3 ml of bacterial culture and 3 ml of soft agar on broth plate. The plates were incubated at 37°C overnight, and number of plaques was counted. RESULTS AND DISCUSSION To deaminate is known

to act

conditions

cytosine specifically

(13,14).

has been

between

bisulfite, uracil

Although treatment

we have

two portions

performed

to

with

of a wild

uracil-DNA

glycosylase

presence

reaction

needed

rates

of high

mild of

cytosine

for

step

1 and 2

concentrations

to convert

uracil a following

2 M sodium

a sample

the alkaline

an extract the

is

; one was dialyzed

As a control,

and exposed

under

deamination

adduct&uracil-

presence

bisulfite-induced

$X174 DNA was incubated at pH 6.

the

which

the

uracil

of adduct

to

3).

To see whether glycosylase,

bisulfite,

derivatives

of bisulfite-induced

pH 5 and 6 in the

an alkaline (step

or its

sodium

cytosine&cytosine-bisulfite

adductauracil.

are optimal

of DNA we used

on cytosine

The process

shown as follows;

bisulfite

into

residues

of EDTA.

type

pH. strain

activity It

is

removed

experiment. bisulfite

against

or of (2).

was found

types

a urg

1313

DNA which

and then

divided

at pH 9 and the with which

treated

is

with

defective

was performed has been

other

2 M NaCl at pH 5.4

of DNAs were

mutant,

The reaction that

Single-stranded

at pH 5.4 a buffer

of DNA was incubated The three

by uracil-DNA

treated

in

at pH 6 in with

BIOCHEMICAL

Vol. 83, No. 4, 1978

bisulfite

and then

by an extract or exposed even

alkali

of TKJ6901 to the

lesions

type

attacked

1A).

pH without

extract

(Fig.

acts

on uracil

glycosylase in DNA and that

the urg

RESEARCH COMMUNICATIONS

by an extract

(urg-l)(Fig.

alkaline

by the wild

uracil-DNA

is

AND BIOPHYSICAL

168T

DNA treated

bisulfite but

This

not

is

but

not

bisulfite

was not

result

other

indeed

(urp+)

with

treatment

1B and C).

mutant

of

alone attacked

indicates

that

bisulfite-induced

defective

in

the

enzyme

activity. The --in vivo role by comparing survivals Phage

SPOZ which

the

survivals in

control

phages,

exposed

to

the

the

DNA of

had been

alkaline

pH in

SP02,

cells that

place

in 3+

even

expectation. cells treated of

TKJ5572

isogenic

strains, polA151)

4 was obtained.

x-

cells

polA

mutations

no effect or irradiated reactivated

higher TKJ5532

with

in x+ (m+,

used. four types

reactivation

in the polA-

It is

is

than

in

with

B.

this and urg-

in urg-

cells.

above

notion.

the

TKJ5573

of

and the other

hosts,

effects

hand,

treated

although Thus, the a+

1314

has been

formed

(urg-1,

polA+),

polA151), which were constructed When SP02 phages treated with

cells, phages

the

anticipated,

the

result

SP02 was less

light, cells.

(16).

DNA (15),

3 accords

cells

On the of

ultraviolet

that

subjected

of bisulfite/alkali-

polA+),

(2),were on the

additive.

in its

to confirm

(urg-1,

as in polA-

were

indicates

PBS1 in urg+

Bisulfite/alkali-treated

as well on the

in Fig.

was performed

9 hr and

bisulfite/alkali-treatedPBSltakes

survivals

and TKJ5581

by genetic transformation various agents were plated Fig.

It

with

involved.

thymine

for

same whereas

considerably

for is

glycosylase

shown

between 12 hr of

pH 5.4

bases

PBS1 (7,17).

reactivation

experiment

(urg+,

of

uracil-DNA

The result the

difference

result

is

of bisulfite/alkali-treated

essentially

An additional

four

in place with

cell

cells.

SP02 were

A set

the normal

to the

increased

was observed

This

function

for

two types

exposure

or cytosine-adducts,

contains

infection

of cells

infectivity

manner.

the urg+

for

times in =+

in 2 M NaCl at

uracil-

uracil

no host Survivals

were

not

an inhibitor

after

therefore,

incubated

on the

a maximum after

in

cells.

at pH 5.4

and the

reached

change

in which

contains

that

subtilis

hosts

a similar

but

which

PBS1 phage

demonstrated

of

which

mechanism

Unlike

decreased,

and x-

plated

increasing phage

was investigated

bisulfite

and then

2, with

rather

two types

uracil,

repair

times

No significant

bisulfite-induced to

in Fig.

in x+

2 M sodium

various

cells

the

treatment.

in DNA repair phages

with

of bisulfite-treated

in x-

survivals

alkaline

treated

As shown

pH survivals

while

glycosylase

of bisulfite-treated

to pH 9 for

cells.

alkaline

uracil-DNA

had been

9 hr was exposed of host

of

of

the urg with

methyl

shown

reactivated

the u mutation

and the exerted

methanesulfonate

these phages were less function is specifically

in in

BIOCHEMICAL

Vol. 83, No. 4, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

2 20 6 Y L s .A! 2.. 10 2 z 8 a a" IO

20

10

20

Fraction

IO

20

number

Fig. 1. Sedimentation in alkaline sucrose of bisulfite-treated DNA after incubation with g. subtilis extracts. (A) DNA treated with bisulfite and alkali. (B) DNA treated with bisulfite alone. (C) DNA treated with alkali. [3H]Thymine-labeled $X174 DNA was treated with 2 M NaHS03 in 0.5 M sodium acetate (pH 5.4) at 37°C for 54 min. After chilling, the DNA sample was divided and dialyzed against three changes of 500 ml of 10 mM TriseHCl-1 mM EDTA (pH 9.0)(for A) or 10 mM Trisamaleate-1 mM EDTA (pH 6.0)(for B) at 0°C For C, DNA was treated with 2 M NaCl in 0.5 M sodium acetate for 18 hr. (pH 5.4) and then with 10 a&l Tris'HCl-1 mM EDTA (pH 9.0) in a similar manner. The DNA samples (O.lpg)wereincubated at 37'C for 20 min with or without an extract of B. subtilis (10 pg protein) in 0.1 ml of 0.1 M Tris.maleate-50 mM EDTA (pH 6.0), and analyzed by alkaline sucrose gradient centrifugation. Incubated with an extract of g. subtilis 168T(urp+)(o); incubated with an extract of TKJ6901 (urg-l)(o); incubated without an extract (x). required

for

the

repair

of bisulfite/alkali-induced

lesions,

namely

uracil,

in DNA. Finally, bacteria into

effect

of x

was examined. 20 ml of

Portions

of

Difco three

each

100 Ug/ml

incubation

at 37°C were per

1O1'

correction

than

are wild

Recently, mutator increased

scored

of

of

type

as well Duncan activity. sensitivity

were Numbers

(uf)

seems

arised

the

uracil

strains. and Weiss We also

Nitrous

acid

and guanine

is known

(20)

found

that

that

a x

to bisulfite

and is 1315

2 days

agar of

of rifampicin-resistant repair

(urg-1)

were

system

works

167 + for

coli mutants defective in sensitive to nitrous acid

residues

found

37°C.

as well.

reported that Escherichia (ung mutants) are more

as adenine

at

on nutrient after

and TKJ6901 that

of

inoculated phase

plated

formed

frequency

were

to stationary

cultures

168T It

spontaneously

cells

and colonies as mutants.

cells

mutation

subtilis

and grown

independent

respectively.

Roza --et al. (18) uracil-DNA glycosylase residues

broth

rifampicin,

viable

49 and 526 f 99, the

of

of

on spontaneous

1 x 10" g.

nutrient

containing mutants

mutation

About

&.

to deaminate of nucleic

coli

mutant unable

ung

mutants

of E. & to reactivate

cytosine

acids

(19). have

a

exhibits phage

an Tl

BIOCHEMICAL

Vol. 83, No. -4, 1978

t Bisulfite treatment

AND BIOPHYSICAL

+/

!

RESEARCH COMMUNICATIONS

-gFe;-

\

0

I

I

10

20

Time

of

incubation

30 ( hr)

Fig. 2. Effect of alkaline treatment on plaque-forming abilities of bisulfitetreated SP02 phages in s+ and ucells. SPO2cl was incubated in 2 M NaHSOs-0.5 M sodium acetate (pH 5.4) or 2 M NaCl-0.5 M sodium acetate (pH 5.4) at 37'C for 9 hr, and then diluted 100 fold with Tris dilution fluid (pH 9.0). The diluted phage suspension was kept at 0°C for various times and plated on cells. Bisulfite-treated SP02 plated on: 168T (urp+) two types of g. subtilis (0); TKJ6901 (urg-l)(o). Non-treated SP02 plated on: 168T (x+)(A); lXJ6901 (w-g-l)(A).

0

3

6

9 lime

of

0 incubation

Survivals of bisulfite-treated SP02 and Fig. 3. strains of B. subtilis. (A) Survivals of SPOZ. SPOZcl or P&l was incubated in 2 M NaHSOs-0.5 M at 37oC. At times indicated, phages were diluted fluid (pH 9.0). After standing at O'C for 12 hr, Plated on B. subtilis 168T (m+)(o); on TKJ6901

1316

1 ( hr)

2

PBS1 in urg+ and urg(B) Survivals of PBSl. Sodium acetate (pH 5.4) 100 fold into Tris dilution phages were plated. (urg-l)(o).

BIOCHEMICAL

Vol. 83, No. 4, 1978

v

L Time

4 of

b incubation

8

0 (hr)

AND BIOPHYSICAL

30

Time

60 90 incubation

of

120 (min

)

RESEARCH COMMUNICATIONS

120 60 of irradiation

0 Time

160 (set

Fig. 4. Effect of urg and ~ mutations on plaque-forming abilities of SP02 phages treated with various agents. (A) Bisulfite treatment: SPO2cl was incubated in 2 M NaHSOa-0.5 M sodium acetate (pH 5.4) at 37'C. At times indicated, phages were diluted 100 fold with Tris dilution fluid (pH 9.0) and kept at 0°C for 12 hr. (B) Methyl methanesulfonate treatment: SPOZcl was incubated with 0.05 M methyl methanesulfonate in M9 medium at 37“C. At times indicated, phages were diluted with M9 medium. (C) Ultraviolet irradiation: SPO2cl was suspended in M9 medium and irradiated for times indicated. The dose rate was approximately 0.9 J/m=. The treated phages were plated on following strains: TKJ5532 (urg+, m+)(o); TKJ5573 (urg-1, polA15l)(r); TKJ5581 (urg-1, polAllil)(h). polA+j (0) ; TKJ5572 (urg+,

pre-exposed with is

the

to bisulfite present

involved

enzyme

in repair

recognizes

the

absence

this

repair

mechanisms

and weak alkali

observations,

of deaminated

uracil

per

se,

of uracil

from

mechanism.

This might the permanent

to prevent

(21).

strongly

cytosine but

DNA as the

These

suggest not

together

uracil-DNA

residues

of DNA.

a mismatched

normal

results,

that

constituent

base

glycosylase

Since the pair (l-9,21),

is prerequisite

be the reason why cells possess inclusion of uracil into DNA.

to

elaborate

ACKNOWLEDGEMENTS: We thank Drs. S. Okubo, H. Tanooka, N. Munakata and F. Makino for bacterial and phage strains used, and Drs. H. Hayatsu, K. Shimizu and T. Horiuchi for discussion. This work was supported in part by research grants from the Ministry of Education, Science, and Culture of Japan.

1. 2. 3. 4. 5.

REFERENCES Tye, B., Nyman, P., Lehman, I. R., Hochhauser, S., and Weiss, B. Proc. Nat. Acad. Sci. USA. 74, 154-157. Makino, F., and Munakata, N. (1977) J. Bacterial. 131, 438-445. Warner, H. R., and Duncan, B. K. (1978) Nature, 272, 32-34. Shapiro, R., and Klein, R. S. (1966) Biochemistry, 5, 2358-2362. Lindahl, T., and Nyberg, B. (1974) Biochemistry, 13, 3405-3410.

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Vol. 83, No. 4, 1978

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Lindahl, T. (1974) Proc. Nat. Acad. Sci. USA. 71, 3649-3653. Friedberg, E. C., Ganesan, A. K., and Minton, K. (1975) J. Virol. 16, 315-321. Sekiguchi, M., Hayakawa, H., Makino, F., Tanaka, K., and Okada, Y. (1976) Biochem. Biophys. Res. Commun. 73, 293-299. Gates, F. T., III, and Linn, S. (1977) J. Biol. Chem. 252, 1647-1653. Lindahl, T. (1976) Nature, 259, 64-66. Sinsheimer, R. L. (1966) Procedures in Nucleic Acid Research, pp. 569576, Harper & Row, New York. Yasuda, S., and Sekiguchi, M. (1970) J. Mol. Biol. 47, 243-255. Hayatsu, H. (1976) Progr. Nucl. Acid Res. Mol. Biol. 16, 75-124. Shapiro, R. (1977) Mutation Res. 39, 149-176. Okubo, S., and Romig, W. R. (1965) J. Mol. Biol. 14, 130-142. Takahashi, I., and Marmur, J. (1963) Nature, 197, 794-795. Tomita, F., and Takahashi, I. (1975) J. Virol. 15, 1073-1080. Roza, R. D., Friedberg, E. C., Duncan, B. K., and Warner, H. R. (1977) Biochemistry, 16, 4934-4939. Schuster,H.,and Schramm, G. (1958) 2. Naturforsch. 13b, 697-704. Duncan, B. K., and Weiss, B. (1978) J. Supramol. Biol. suppl. 2, 56. Hayakawa, H., Kumura, K., and Sekiguchi, M. (1978) J. Biochem. (in press).

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