Genetic effects of halogenated thymidine analogs incorporated during thymidylate synthetase inhibition

Vol. 2, No. 6

BIOCHEMICAL

GENETIC EFFECTS OF HALOGENATED INCORPORATED DURING THYMIDYLATE Z. Institute

Received

are

thymine

but was

shown

analogs

or by interference

of prototrophic

either with

and 5-

acid

and Griboff,

(DNA)

1954;

Dunn

strains

was

of

and Smith, negligible,

by the use of deoxyribosylated

the endogenous

or sulfanilamide

purpose

(Zamenhof

of the present

of the incorporation the DNA

methylation

of deoxyuridylic

et al.,

1958;

To create deoxyuridine This Fellow General Poland.

Dunn

and

the consequences

(SM)

dependent,

thymidine-non-requiring

used

extensively

in our

earlier

(IUDR)

into

strain

mutagenicity

studies

1958). artificially

(FUDR) research

was to evaluate and 5-iododeoxyuridine

coli,

and Szybalski,

studies

of 5-bromo-(BUDR)

of streptomycin

Sd4 of Escherichia

**;*

the deoxyribonucleic

the DNA

to be increased

5-bromo-,

1957). The

*e

5-chloro-,

(Zamenhof

into

by aminopterin

0

into

bacteria

Incorporation

(Iyer

analogs,

incorporated

1957).

Smith,

University,

1960

thymine-deficient

acid

ANALOGS INHIBITION*

and W. Szybalski**”

Lorkiewicz””

Halogenated iodouracil,

THYMIDINE SYNTHETASE

of Microbiology, Rutgers, The State New Brunswick, New Jersey

May 19,

June 1960

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was was

conditions employed,

supported

of the Rockefeller Microbiology,

of thymidine an analog

in part

deficiency,

5-fluoro-

to inhibit

irreversibly

known

by the National

Science

Foundation.

Foundation; on leave from Department of University of M. Curie-SHodowska, Lublin,

Present address: McArdle Memorial Wisconsin, Madison 6, Wisconsin. 413

Laboratory,

University

of

Vol. 2, No. 6

BIOCHEMICAL

thymidylate

synthetase

FUDR

alone

partially growing

(0.25-0.50

cells

Na2HP04; 30 200

were

or

ml

IUDR

Under

these

the

in

bacterial

thymidine the

Figure

1.

Inhibition

FUDR

(0.5

pg/ml)

reversal

by

pg FUDR/ml) curves

l

) and

E.

its

(200

pg

BUDR/ml

conditions

mutants

were

grown

cells.

strain

are

and

observed

susceptible

propagated

for

51%

and

two

IUDR

of FUDR,

to

three

43%

of

substitution

respectively.

kg/ml)

cultures.

of

rates

toward

(Iyer

and in

FUDR-, controlling

to

supplemented

growth

increases

loci

19%

casamino

water)

absence

g

+ 0.5

among the

the

is

0.021

vitamin-free

replaced

which

Logarithmically

g KH2P04;

dist.

and

effect

by

cells

earlier

1).

1958).

partial

) control

two-fold

Thus not

15.6%

IUDR-labeled

described

ml

June 1960

et al., an

(0.01

FUDR,

In

with

mutation

(Fig.

3 g Bacto

BUDR

(200

( 0

ml

1000

DNA.

Sd4

) and

higher

methods

5 mg

( A

BUDR-free

significantly

and

coli

together

or

CaC12;

Cohen growth,

SS medium

1M

of only

of

per

conditions

BUDR-supplemented

BUDR-

some

extent

( W ),

for

by

the

BUDR

BUDR

3 g NH4Cl;

0.3

1957;

bacterial

into

g MgS04.7H20;

BUDR

to

kg

transferred

generations.

occurred

inhibits

50-200

g glycerol; mg

et al.,

pg/ml) by

0.3

acids;

(

(Heidelberger

reversed

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

mutagenic 414

E.

coli

Sd4

do

not

generally

exhibit

SM

independence,

as

assessed

Szybalski,

1958),

although

under

the

frequency

of SM-independent

FUDR

+ BUDR-,

mutation

to

effect

SM

or

FUDR

independence

of BUDR

or

IUDR

+ IUDRin to

this the

Vol. 2, No. 6

same

BIOCHEMICAL

extent

as

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

certain

loci

in

phage

1959)

(Freese,

or bacteria

June 1960

(Zamenhof,

1959).

observed these

Although

no

with

BUDR

analogs

process. grown

clearly or

affect It was

bacteria

in

perceptible IUDR,

any

reported

it was

way by

become

direct

the

interesting

and

light Zamenhof

sensitive

to

UV

effect

to determine

ultraviolet

Greer

highly

mutagenic

was whether

(UV)-initiated (1957)

light.

mutagenic

that

The

latter

5-bromouracileffect

was

C A 0.ooo10

SXIO~ UV

t

2x10’

IX@

DOSE

DOSE INCAEASE RATIO IPHOloRLAcllvATIONI

I pJ4 5 DOSE REDUCTION RATIO (RAoI~SENS~T~~A~~~N)

3X@

(ERGS/mm=)

Figure 2. A: UV survival before (solid lines) and after photoreactivation (broken lines) of E. coli Sd4 grown in the absence (control) or in the presence of BUDR or IUDR (ZOO pg/ml) on a SM (20 pg/ml) and FUDR (0.5 pg/ml) supplemented medium. B: Graphic comparison of photoreactivability for the control and for the BUDREach point represents a UV dose ratio for identical survivals grown cultures. of the photoreactivated and non-reactivated cells, as determined from Figure C: UV sensitization of BUDRand IUDR-grown cells. Each point represents UV dose ratio for identical survivals of the control and of the BUDRor IUDRsupplemented cultures.

clearly or

IUDR

(2O”C,

confirmed (Fig. saline

with 2).

and

suspension).

E.

coli

irradiated

Sd4

grown with

The

in

the

presence

a Westinghouse

resulting 415

radiosensitization,

of FUDR Sterilamp

and

BUDR

G15T18 expressed

as

2A. a

Vol. 2, No. 6

BIOCHEMICAL

the UV dose

ratio

(dose

for

reduction

identical

ratio),

substitution)

or 2.2

essentially

cells

Figure

(Fig.

3.

mutants

(43 mol

the absence pg/ml)

of UV light

and analog-grown

as 4.7

substitution)

was

related

observed

(50 mol

(Fig.

% BUDR

Xc).

to its bactericidal

between

cells

normal

effect,

and analog-labeled

c: > 3 In -9 Q: 103

of UV-induced Sd4 cultures

or presence

grown

of FUDR

or in combination

(200 pg/ml),

% IUDR

June 1960

3).

coli

alone

of normal

to as much

no differences

Frequency

in E.

survivals

amounted

The mutagenicity with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and plotted

ii! e

in

5 $102

(0.5

with

BUDR

as a function

s E ~ IO’

of

UV survival. f 2 loo

While

attempting

photoreactivated less

to determine cells,

photoreactivable

tubes; ment

8 cm distance; with

inactivated

effect

was (2 hr,

found

that

37 “C ) than

37”C,

coli

10 pg TEM

of mutants

BUDR-labeled

in saline; normal

among cells

2 fluorescent cells

2A,

nor

the BUDR-labeled

cells

under

the conditions

employed.

of other

Sd4 cells per

incorporation agents

treated

with

ml of distilled 416

and Stahl

ZB),

the normal

and IUDR

on radiation known

to modify

triethylenemelamine water,

followed

slightly

20 Watt

(1960)

light

and Gkun

(Fig.

are

of Greer

the effect E.

that

2 hr;

fluorescent

of BUDR

us to investigate

observed

suspended

Neither by white

the frequency

(cells

the observations

communication).

The

it was

IO

in agree-

(private were

sensitivity DNA

prompter

structure.

It

(TEM) by 2 hr growth

in

Vol. 2, No. 6 nutrient

BIOCHEMICAL

medium),

of chemically Szybalski,

a powerful

modifying 1960),

presumptive gift

become

J. Lein,

Bristol

hr (37’C)

in SS medium

The

are

more

modified

DNA

and Doty, BUDR with

between molecule

1959) was

chloroform

was

formed

in a Beckman

(0.15M

NaCl,

the “melting and 89.O”C,

BUDR

of the

temperature

cells,

The equipped

of normal

it was greatly

found

(Marmur DNA

with

RNase

measurement, with

(51%

deproteinized

per-

thermospacers

no significant

difference

and BUDR-labeled

increases

and LUDR into

that the thymidylate the incorporation

the DNA

has essentially

no mutagenic

effect

the cells

UV-sensitive

and less

highly

for

temperatures.

lability

of RNA by digestion

revealed

” temperatures

slight

broth.

and BUDR-labeled

precipitation.

citrate),

effect

ml;

compounds

of elevated

Duponal-lysed

DU spectrophotometer sodium

pg per

DNA

respectively).

In summary,

analogs

and alcohol

0.015M

FUDR,

for normal

and freed

and

5-bromouracil-substituted

The melting

from

Y.)

to these

and the heat

examined.

determined

(0.1

effect

phenomenon

and butanol,

by dialysis

inhibitor,

this

mitomycin N.

of E.

coli

on the Sd locus, photoreactivable.

417

synthetase of the thymidine

Sd4.

This although

capable

survival

and another

10% nutrient

that

to the lethal

extracted

followed

(91.5

(1960)

lower

pg per ml)

exposed with

agent

(Lorkiewicz

(lo-fold

Syracuse,

of cells

by Greer

incorporation),

between

Laboratories,

sensitive

relationship

(0.5

inhibitor,

supplemented

reported

of DNA

to UV light

FUDR

UV sensitivity

and an alkylating

components

synthetase

in the

It was

mutagen

sensitive

Similarly,

increases

bacteria

more

thymidilate

of Dr.

bacterial

the pyrimidine

at 3 x IO3 ergs/mm2).

Juno 1960

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

substitution it renders

2

Vol. 2, No. 6

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Acknowledgments: helpful

discussions

We are and to Miss

indebted

Clara

to Dr.

Bird

A.

W.

June 1960

Kosinski

for the skillful

for

assistance.

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