BIOCHEMICAL

Vol. 71, No. 1, 1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REGULATION OF FUNGAL RIBONUCLEOTIDE REDUCTASE BY UNUSUAL DINUCLEOTIDES1 William

H. Lewis2,

David

Department

R. McNaughton3, of Microbiology,

Winnipeg,

Received

April

22,

Herb

B. LGJohn

University

Manitoba,

Canada

and Jim A. Wright

of Manitoba

R3T 2N2

1976

SUMMARY: Three phosphorylated dinucleosides designated HSl, HS2, and HS3, isolated from the water-mould Achlya, were shown to significantly inhibit riboAll three compounds decreased CDP nucleotide reductase activity from Achlya. reduction in fungal extracts by 50% at concentrations of O.lmM. At the same concentration HS3 also inhibited partially purified CDP reductase from Chinese hamster ovary cells by at least SO% but showed only 10% inhibition with enzyme from E. coli. ADP reductase activity from Achlya was inhibited 50% by both HSl and HS3 at O.lmM. HS2 however, showed no inhibitory effect on purine reduction. The levels of ribonucleotide reductase during the asexual growth cycle of Achlya correlated with thymidine uptake into DNA and with the synthesis of HS compounds. Recently, phosphorylated thesis with

L&John --et dinucleoside

in the water-mould the various

determine

DNA synthesis.

the

the replication

specific

stages

been

suggested

in this for

(3).

the production

known side

process

compounds+

to be subject

the

is known of their

of the

to complex rate-limiting

Thus this

enzyme seemed a likely

step

a role

by which

periods

(2).

syn-

We wished

in the eucaryotic

is

often

regulation

to of

organisms confined

to

cycle.

It

of the cell

may be a major

regulation

triphosphates

may be the

protein

point

has

of control

systems this enzyme is responsible of the four deoxyribonucleotides and is

allosteric

reduction

active

manner

DNA synthesis

reductase

highly

have been shown to interact

play

process

DNA.

In other eucaryotic of a balanced supply

and deoxyribonucleoside

compounds

may also

of three

during

in a complex

and specific

ribonucleotide

isolation

synthesized

These

same compounds

of development that

reported

RNA polymerases

Little

regulate

(1)

Achlya.

fungal

whether

al

(4).

by a variety Furthermore,

in DNA synthesis

candidate

for

of ribonucleo-

ribonucleotide

and cell

division

(5).

HS regulation.

Supported by grants from The National Research Council of Canada to H.B.L. and J.A.W. 'National Cancer Institute of Canada Studentship Recipient. 3National Research Council of Canada Scholarship Recipient. +A revised report being prepared (McNaughton, D.R., Klassen, G.K. and L&John, H.B.) will show that contrary to the structures proposed in a preliminary communication (l), HS3 dinucleoside polyphosphate contains uridine and adenosine that are highly modified. Both HS2 and HSl dinucleoside phosphates contain only uridine that is also modified. The modifying components on the uridine moities absorb UV.

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

128

BIOCHEMICAL

Vol. 71, No. 1,1976

Ribonucleotide organisms there

but

have been

duction

resembles

while

that

bles dine

only

in fungal

(6)

the

reductase

has not

extracts.

second

class here

ribonucleotide

systems. trations

coli

of all

similarly

inhibited

of the three

ribonucleotide MATERIALS

activity enzyme

to require

found

coenzyme found of both

resembling

those

significantly

dinucleotide

compounds, but

is

showed

compounds,

three

compounds.

also

inhibited

only

in Lactobacillus purine found

while

effect

resem(8).

and pyrimiin mammalian by low

concen-

ADP reduction

HS3, the most CDP reduction

marginal

properties,

B12 and thus

inhibited

re-

in Saccharomyces

in general

reductase

activites

of eucaryotic

To our knowledge,

ribonucleotide

in Achlya

by two of the

reductase

pyrimidine

the demonstration

unusual

inhibitor

a variety any.

type

appears

CDP reduction

three

from from

of ribonucleotide reductase

Furthermore,

isolated

The CDP reductase (7)

RESEARCH COMMUNICATIONS

purified

demonstrating

and mammalian

Pythomyces

We report

has been

significantly

two reports

the -.E from

been

AND BIOPHYSICAL

is

potent

by mammalian

on enzyme from E. &.

& METHODS

Organisms. The conditions for growing of cells have been described (9,lO). E. coli strain, was obtained from the E. Coli Genetic ovary cells (11) were routinely maintained in (Flow Laboratories Inc.) as previously described

Achlya sp. (1969) and harvesting KL161, a thymine requiring Stock Centre. Chinese hamster a-minimal essential medium (12).

Preparation of cell extracts. Routinely, Achlya cells were harvested by filtration 10 hours after spore germination. The cells were ground to a fine powder in liquid nitrogen and 0.5 ml of buffer (O.lM Hepes* pH=8, 5.OmM dithiothreitol) added for every gram of cell powder. The extract was centrifuged at 50,OOOg for 20 min and the supernatant was then passed over a column of Sephadex G25 (1 X 16 cm, equilibrated with 5OmM Hepes pH=7.5, 5.OmM dithiothreitol). The extract was then frozen in liquid nitrogen and stored at -76'C. Protein content of the extracts was measured by the method of Lowry --et al (13). E. coli was grown and crude extract prepared as described (14). The extract was dialysed 4 hours against 50mM Hepes pH=7.5, 5.OmM dithiothreitol. Chinese hamster ovary cell crude extract was prepared as described (15). The mammalian enzyme was partially purified by precipitation at pH=5.2, followed by affinity chromatography on ATP-agarose (Lewis and Wright, manuscript in preparation). Enzyme assay. CDP reductase activity was routinely measured in a reaction mixture4of 5OmM Hepes pH=7.5, 1OmM MgCl,, 1OmM dithiothreitol, 1OmM ATP volume of 150 pl. After incubaand 0.4mM d C)CDP (4000 cpm/ nmole) in a total tion at 22 C for 60 minutes the reaction was terminated by heating in boiling water. The deoxycytidine formed was measured as previously described (15). ADP reductase was routinely measured in a reagtion mixture of 50mM Hepes l.OmM dGTP and 0.4mM ( C)ADP (4300 cpm/nmole) in pH=7.5, 1OmM dithiothreitol, The deoxyadenosine formed was measured as described a total volume of 150 pl. by Cory (16).

* Abbreviations:

Hepes = N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic TEAB = triethylammonium bicarbonate

129

acid,

BIOCHEMICAL

Vol. 71, No. 1,1976

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Thymidine incorporation. Thymidine uptake into acid precipitable material was measured by adding ['Hjthymidine (0.4 uCi/ml, 20nM) to 25 ml of Achlya culture at various times after spore germination. After 10 minutes incubation, cells were filtered, washed with phosphate buffered saline and incubated in icecold 10% trichloroacetic acid for 10 minutes. The cells were then filtered onto a glass fibre filter (Gelman 161630) and treated as previously described (15). 32 P-orthophosphate pulsing. Twenty-five ml sgyples of cells were removed at the intervals indicated and pulsed with 200 uCi i Plorthophosnhate for 10 min. The labelled cells were washed with water'by vacu;m filtration and extracted for 30 min with 0.2 ml of l.OM formic acid at O'C. HS compounds in the acid extracts were separated on PEI-cellulose plates (Brinkman) and the area corresponding to HS3 cut and radioactivity determined. Isolation and purification of HS compounds. Mid-log phase cells of Achlya (14 hr. old) grown and harvested as described (2) were used for extraction of HS compounds. Typically, 50-608 wet weight of cells were extracted with 50 ml of 1M formic acid, pH=3.5, at 0 C for 1 hr. The acid extract collected by filtration was carefully adjusted to pH=7 with NaOH and then diluted 10 fold. All subsequent purification steps were performed at 4'C. After dilution,22 ml of a formic acid extract from cells grown in medium supplemented with ( P)orthophosphate was added and the entire volume was then flushed through a column (3X45 cm) of DEAE-Sephadex A-25 at a flow rate of 50 mllhr. The column was pre-equilibrated with 0.2M TEAB buffer at pH=7. The loaded column was then washed with 500 ml of 0.2M TEAB before nucleotides were eluted with a linear gradient (0.2M - 1.2M) of TEAB at pH=8. The absorbance of each fraction was determined and fractions corresponding to peaks of UV absorbance assayed by chromatography on PEI-cellulose for HS content as described (1). The three HS compounds eluted at the same molarity of l.lM TEAB. The pertinent fractions were pooled, diluted 10 fold and loaded onto another DEAB-Sephadex A-25 column (1X30 cm) pre-equilibrated with O.lM NaCl:O.O5M Na formate, pH=3.6 buffer. After loading, the column was washed with the NaCl-formate buffer and nucleotides eluted with a linear gradient of NaCl (O.lM - 0.6M) in 0.05M Na formate, pH=3.6 buffer. The three HS compounds eluted at NaCl concentrations as follows: HS3, 0.28M; HS2, 0.35M; and HSl, 0.4OM. ATP marker eluted at 0.22M. The HS fractions were pooled according to species, concentrated to 1 ml by evapomix and desalted on Sephadex GlO columns using 0.04M TEAB, pH=7 as elution buffer. The desalted HS solutions were brought to dryness by evapomix, washed six times with successive additions of 1 ml water. The final residue was dissolved in 1 ml of water and stored at -2OOC until use. Chemicals. Radioactive thymidine, CDP and ADP were purchased from AmershamlSearle. ATP-agarose was prepared by PL Biochemicals. Sephadex GlO, G25 and DEAE-Sephadex A-25 were obtained from Pharmacia Ltd. All remaining chemicals were obtained from Sigma Chemical Co. RESULTS Ribonucleotide extracts

of Achlya

reductase cells

in Achlya.

harvested

10 hours

When prepared as described, crude after spore germination contained

at least 20 mg of protein move endogenous inhibitors

per ml. After passage through of low molecular weight, both

ribonucleotide

were

with

protein

had an optimum

reductions concentrations temperature

Sephadex G25 to repurine and pyrimidine

demonstrated.

above of 22'C

Both CDP and ADP reductase systems (17). Table 1 shows that

The activity increased linearly mg protein per assay. The reaction and proceeded linearly for at least 60 min.

0.75

activities resembled those CDP reduction in Achlya is

130

found in mammalian dependent upon

BIOCHEMICAL

Vol. 71, No. 1,1976

AND BIOPHYSICAL

PROPERTIES OF RIBONUCLEOTIDE

TABLE 1

RESEARCH COMMUNICATIONS

REDUCTASE FROM ACHLYA

CDP REDUCTASE UNITS*

OMISSION OR ADDITION complete

1.92

- ATP

0.28

ADP REDUCTASE UNITS 0.77

- dGTP

0.01

+ dATP (1.0 + Hydroxyurea

mM) (1.0

+ Fe(NH4)2(S04)2

mM) (0.1

mM)

0.21

0.08

0.24

0.36

1.95

0.63

-

* 1 unit formed

of ribonucleotide per

hour

reductase

per mg extract

= 1 nmole

deoxynucleoside

protein.

IO -

a.

0.02 HS

Figure

O-04 0.06 COMPOUND (mM)

0 06

b.

0 IO

Cl.02 004 006 006 HS eOMPO”ND(mM1

0 I(

1. The inhibition of Achlya ribonucleotide reductase activity by HS compounds. CDP reductase (Fig. la) and ADP reductase (Fig. lb) were assayed as described in Materials and Methods, with 0.80 mg extract protein per assay. Each point for HS3 (u) represents the from three separate experiments average of duplicate points utilizing two separate preparations of HS3 compound and cell extract. The points for HS2

Regulation of fungal ribonucleotide reductase by unusual dinucleotides.

BIOCHEMICAL Vol. 71, No. 1, 1976 AND BIOPHYSICAL RESEARCH COMMUNICATIONS REGULATION OF FUNGAL RIBONUCLEOTIDE REDUCTASE BY UNUSUAL DINUCLEOTIDES1 Wi...
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