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