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J. Biol. Chem., Vol. 277, Issue 21, 18574-18578, May 24, 2002
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From the Department of Medical Biochemistry and Biophysics, Umeå
University, SE-901 87 Umeå, Sweden
Received for publication, February 15, 2002
The ribonucleotide reductase system in
Saccharomyces cerevisiae includes four genes
(RNR1 and RNR3 encoding the large subunit and
RNR2 and RNR4 encoding the small subunit).
RNR3 expression, nearly undetectable during normal growth,
is strongly induced by DNA damage. Yet an rnr3 null mutant
has no obvious phenotype even under DNA damaging conditions, and the
contribution of RNR3 to ribonucleotide reduction is not
clear. To investigate the role of RNR3 we expressed and
characterized the Rnr3 protein. The in vitro activity of
Rnr3 was less than 1% of the Rnr1 activity. However, a strong
synergism between Rnr3 and Rnr1 was observed, most clearly demonstrated
in experiments with the catalytically inactive Rnr1-C428A mutant, which
increased the endogenous activity of Rnr3 by at least 10-fold. In
vivo, the levels of Rnr3 after DNA damage never reached more than
one-tenth of the Rnr1 levels. We propose that heterodimerization of
Rnr3 with Rnr1 facilitates the recruitment of Rnr3 to the
ribonucleotide reductase holoenzyme, which may be important when Rnr1
is limiting for dNTP production. In complex with inactive Rnr1-C428A,
the activity of Rnr3 is controlled by effector binding to Rnr1-C428A.
This result indicates cross-talk between the Rnr1 and Rnr3
polypeptides of the large subunit.
DNA damage in eukaryotic cells leads to arrest of the cell cycle
and activation of the genes involved in DNA repair (1). The DNA damage
checkpoint pathway responsible for activation of DNA damage-inducible
genes in yeast Saccharomyces cerevisiae has been the focus
of intensive research during the past decade (2). The emerging
conservation between the DNA damage checkpoint pathway in yeast and
humans promotes a better understanding of this vital process (3, 4).
One of the frequently used tools in the dissection of the DNA damage
checkpoint pathway in S. cerevisiae has been the gene
RNR3 (5). This gene is induced 5-10-fold (6-8), and in
some reports more than 100-fold (5), in response to DNA damage. In
addition, it is not essential, and the rnr3 null mutation
has no phenotype under all studied conditions (5). These properties of
the RNR3 have been exploited in the identification of a
number of important genes involved in the DNA damage checkpoint function such as CRT1, TUP1, SSN6, and
DUN1 (9-12). On the basis of homology with the
RNR1 gene encoding the large subunit of yeast ribonucleotide
reductase (RNR),1 it has been
proposed that RNR3 encodes a second large subunit of the
yeast RNR (5). In support of this hypothesis, it was demonstrated that
overexpression of RNR3 could rescue rnr1 null mutants (5).
Ribonucleotide reductase is the rate-limiting enzyme in DNA precursor
biosynthesis present in all living cells (13). All RNRs use free
radical chemistry to reduce ribonucleotides to deoxyribonucleotides. Based on the nature of the cofactor providing the free radical for the
reaction, RNRs were divided into three classes (14). Nearly all
eukaryotes have an RNR belonging to class I in which the enzyme is
thought to be an In addition to allosteric regulation, the activity of yeast RNR is
controlled by binding of a protein inhibitor, Sml1, to the large RNR
subunit (16, 17). The Sml1-dependent mechanism is so far
unique for the yeast RNR. During S phase and after DNA damage,
decreased Sml1 levels result in derepression of the RNR activity (18,
19). The decrease of Sml1 levels is caused by post-transcriptional
regulation and requires Mec1/Rad53-dependent phosphorylation. Mec1 and Rad53 protein kinases are essential proteins
and central players in DNA damage response in yeast (2). Deletion of
SML1 rescues the lethality of a mec1 or
rad53 strain, and removal of Sml1 during the S phase defines
the essential function of Mec1 and Rad53 proteins (19).
The S. cerevisiae RNR system consists of the
following four RNR genes: RNR1 and RNR3, which
encode the polypeptides of the large subunit, and RNR2 and
RNR4, which encode the polypeptides of the small subunit
(20-22). The RNR genes are located on different chromosomes (V, IX, X,
and VII, respectively), and expression of all four genes is induced by
DNA damage (11). We have demonstrated before that, in contrast to other
class I RNRs the active form of the yeast small subunit is a We wanted to understand the role of DNA damage-inducible Rnr3 in dNTP
metabolism of yeast. In this paper we present an in vivo and
in vitro characterization of the Rnr3 protein. After DNA
damage, the levels of highly induced Rnr3 never reached more than
one-tenth of the Rnr1 levels, which increased ~2-fold. The Rnr3
protein showed less than 1% of the activity of the Rnr1 in an assay
with the Rnr2/Rnr4 heterodimer. However, a strong synergism between
Rnr1 and Rnr3 was observed (most clearly demonstrated in assays in
which Rnr3 was allowed to form a complex with a catalytically inactive
form of Rnr1). Interestingly, whereas the Rnr1-catalyzed CDP reduction
was inhibited by the allosteric inhibitor dATP, the CDP reduction
catalyzed by Rnr3 was stimulated by dATP. The reaction catalyzed by
Rnr3 together with catalytically inactive Rnr1 showed an intermediate
sensitivity to dATP inhibition, indicating cross-talk between the
allosteric sites in Rnr1 and the catalytic site in Rnr3.
Protein Expression and Purification--
Rnr1 and the
His6-Rnr2/Rnr4 heterodimer were expressed in
Escherichia coli BL21(DE3) using the pET expression vector
(Novagen) as described previously (23). Rnr1 was purified by ammonium sulfate fractionation and affinity chromatography on dATP-Sepharose as
described (24). The co-expressed His6-Rnr2/Rnr4 heterodimer was purified on a nickel-nitrilotriacetic acid-agarose column (Qiagen, Valencia, CA) followed by chromatography on a Bioscale Q10
column (Bio-Rad) (23). Rnr3 was purified from the rnr1
deletion strain Y609 (25) (MATa trp1-1 ura3-1
his3-11,15 leu2-3,112 ade2-1 can1-100 RNR Assay--
Rnr1, Rnr1-C428A, Rnr3, and mixtures of these
proteins were assayed in the presence of pure Rnr2/Rnr4 heterodimer as
described (23).
Sucrose Gradient Centrifugation--
Sedimentation of Rnr1 or
Rnr3 in the absence or presence of dTTP or ATP in a 5-20% linear
gradient of sucrose was performed as described previously (23). All
gradients were calibrated by the addition of catalase
(s20,w = 11.4 S).
Determination of the Levels of Rnr1 and Rnr3 in Yeast Cells Grown
in the Absence or in the Presence of 4-Nitroquinoline-1-oxide
(4-NQO)--
Rabbit polyclonal antibodies were made against the
peptides EKAAPIVDDEET(C) (Rnr1 from amino acid residues 852-863) and
TETIKEDSDEKKC (Rnr3 from amino acid residues 850-862) by Sigma. The
anti-Rnr1 antibodies did not cross-react with Rnr3 or vice versa (data
not shown). The yeast strain W1588-4C (MATa ade2-1
can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1) (the W303
background) was grown in YPDA at 30 °C. Treatment of yeast
with 0.25 mg/liter 4-NQO was started at a density of 1 × 107 cells/ml. After a 3-h incubation with or without drug,
3.6 × 1010 cells were collected by centrifugation and
washed in ice-cold 50 mM Tris-HCl, pH 7.6, 2 mM
dithiothreitol, and protease inhibitors (1 mM
phenylmethylsulfonyl fluoride, 2 mM pepstatin, 0.6 mM leupeptin, and 2 mM benzamidine). The cells
were then suspended in 20 ml of the same buffer and disrupted in a
Bead-Beater. Cell debris was removed by centrifugation (30,000 × g for 20 min followed by ultracentrifugation at 200,000 × g for 2 h). The total amount of protein as
determined by the Bradford assay was ~74 mg. After electrophoresis in
an 8% polyacrylamide gel, immunoblotting was performed basically as
described (26) with the specific anti-Rnr1 or anti-Rnr3 antibodies. The
immunocomplexes were detected by incubation with goat anti-rabbit
antibodies conjugated with alkaline phosphatase followed by
visualization with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium.
DNA Damage Induction of the Rnr1 and Rnr3 Proteins--
To
understand better the contribution of Rnr3 to ribonucleotide reduction,
we estimated the levels of the Rnr1 and Rnr3 proteins in yeast before
and after DNA damage using Western blotting with specific Rnr1 and Rnr3
antipeptide polyclonal antibodies. The expression of RNR genes was
induced by a 3-h incubation of a yeast culture in YPDA medium
containing 4-NQO, and then samples were withdrawn for analyses and
compared with samples from a yeast culture grown without drug. As seen
in Fig. 1, the level of Rnr1 increased
from about 50 to 100 ng of protein per 37 µg of yeast protein extract
(equivalent to 1.8 × 107 cells). There was a
pronounced increase also in the level of Rnr3, from undetectable levels
in the uninduced cells to about 10 ng of protein per 37 µg of yeast
protein extract in induced cells.
Yeast Rnr3 Has a Very Low Specific Activity Compared with
Rnr1--
Rnr3 was purified from the rnr1 deletion strain
Y609 (25) containing a 2-µ plasmid with the RNR3 gene
under a strong and constitutive glyceraldehyde 3-phosphate
dehydrogenase promoter. Using this strain ensured a complete absence of
endogenous Rnr1 contamination and resulted in 1.5 mg of highly purified
Rnr3 per liter of yeast culture (Fig. 2).
The activity of Rnr3 in the presence of an excess of Rnr2/Rnr4
heterodimer was highly dependent on the concentration of Rnr3 (Fig.
3). However, the specific activity of the
Rnr3 even when assayed at 0.5 mg/ml, which is a very high concentration
for an RNR assay, was only approximately 1% of the specific activity
of Rnr1 assayed at a similar concentration (data not shown). The
addition of increasing amounts of Rnr3 to a series of RNR assay
mixtures all containing a constant amount of Rnr1 and an excess of
Rnr2/Rnr4 heterodimer demonstrated a clear synergism between Rnr1 and
Rnr3 in ribonucleotide reduction (Fig. 3). Total RNR activity in these
assays was up to 58% higher than the sum of the Rnr1 and Rnr3
activities assayed separately. This synergistic effect was most
pronounced in assay mixtures with a very low concentration of Rnr1 and
a high concentration of Rnr2/Rnr4 heterodimer.
Synergism between Rnr1 and Rnr3--
The results presented above
suggest that in a complex with Rnr1, Rnr3 has a much higher specific
activity than when it is assayed alone. To address the contribution of
the Rnr1/Rnr3 interaction in the stimulation of Rnr3 activity and
exclude the contribution of Rnr1 activity, we decided to create and use
a catalytically inactive form of Rnr1. We replaced cysteine 428 (corresponding to the catalytically essential cysteine 439 in the
E. coli R1 protein (27)) with an alanine in the recombinant
Rnr1 (Fig. 1). Rnr1-C428A was totally inactive when assayed in the
presence of Rnr2/Rnr4 heterodimer (Fig.
4A); however, the addition of
increasing amounts of the inactive Rnr1-C428A to assay mixtures
containing a constant amount of Rnr3 and Rnr2p/Rnr4 heterodimer
resulted in a pronounced stimulation of RNR activity (Fig. 4). Maximal Rnr3 specific activity was obtained when the ratio of Rnr1-C428A/Rnr3 was approximately 7:1, which gave a value of 11 nmol/min per mg of
Rnr3. This specific activity is approximately 5% of the maximal specific activity of wild-type Rnr1. Surprisingly, no inhibition of
Rnr3 activity was observed with higher amounts of Rnr1-C428A. These
data show that the presence of catalytically inactive Rnr1-C428A increases the endogenous activity of the Rnr3 by at least 10-fold. We
therefore propose that Rnr3 alone has a poor ability to form an active
complex with the Rnr2/Rnr4 heterodimer.
Interestingly, the addition of increasing amounts of Rnr1-C428A to
Rnr1-containing assay mixtures resulted in a slight stimulation of RNR
activity and almost no inhibition at up to 34× molar excess of
Rnr1-C428A over Rnr1 (Fig. 4B).
Rnr3 Has a Lower Temperature Optimum than Rnr1 in the RNR
Assay--
We compared the temperature optimum for reactions catalyzed
by Rnr1, Rnr3, or the Rnr1-C428A/Rnr3 mixture in the presence of the
small subunit (Fig. 5). There was a
distinct difference between the various RNR complexes with the Rnr1
showing the highest temperature optimum followed by the Rnr1-C428A/Rnr3
complex and the Rnr3. These data support the notion that the
Rnr1-containing RNR complex is the most stable followed in stability by
the Rnr1-C428A/Rnr3- and Rnr3-containing RNRs.
Cross-talk between the Allosteric Activity Site in Rnr1-C428A and
the Catalytic Site in Rnr3--
It was shown earlier for the mouse RNR
complex that allosteric effectors strongly influence subunit
interaction (28). Reduction of CDP by yeast RNR containing the Rnr1 was
stimulated by ATP with maximal activity around 5 mM ATP
(Fig. 6A). The optimal ATP concentration for CDP reduction catalyzed by RNR containing Rnr3 was
15-20 mM, whereas the optimal ATP concentration for RNR
containing Rnr3/Rnr1-C428A was the same as observed for Rnr1 alone
(i.e. approximately 5 mM (Fig. 6A)).
The Rnr1-catalyzed CDP reduction in the presence of 5 mM
ATP was inhibited by dATP at concentrations above 50 µM,
whereas the CDP reduction catalyzed by Rnr3 was stimulated by dATP
instead. Interestingly, the CDP reduction catalyzed by Rnr3/Rnr1-C428A
showed dATP inhibition typical for Rnr1 despite the fact that the
catalytic site resides on Rnr3 (Fig. 6B). This result,
together with the ATP optima, indicates cross-talk between the
allosteric activity site on the inactive Rnr1 and the catalytic site on
Rnr3.
Allosteric Effector-induced Oligomerization Is Different for Rnr1
and Rnr3--
When analyzed by sucrose gradient centrifugation, Rnr1
without allosteric effectors sedimented as a mixture of monomers and dimers (Fig. 7A). Upon
addition of dTTP, the sedimentation pattern was dominated instead by
dimers and tetramers. Finally, in the presence of ATP most Rnr1
sedimented as tetramers with a pronounced tailing. In contrast, Rnr3
alone sedimented as monomers, and the addition of dTTP induced only a
minor dimer peak. However, the most pronounced difference was observed
in the presence of ATP where Rnr3 sedimented as an unresolved mixture
of monomers and dimers with no significant tetramer peak (Fig.
7B).
The low levels of Rnr3 protein in yeast cells even after DNA
damage induction, in combination with the very low catalytic activity
of a complex between Rnr3 and the Rnr2/Rnr4 heterodimer, explain why an
rnr3 null mutant has no phenotype under laboratory conditions. Is Rnr3 a redundant protein that has no function in yeast
metabolism? Why is it induced strongly in response to DNA damage? The
complex between Rnr3 and Rnr1 has a much higher catalytic activity than
Rnr3 alone. The presence of such a complex in vivo is
observed in a two-hybrid assay (18). If the levels of Rnr1 are limiting
for ribonucleotide reduction during DNA damage, the expression of Rnr3
may increase total RNR activity. Furthermore, the resistance of
Rnr3-catalyzed ribonucleotide reduction to dATP inhibition might confer
a selective advantage for yeast growing in natural ecological niches
under permanent DNA-damaging conditions.
Why is the catalytic activity of RNR containing Rnr3 so much lower than
the activity of RNR-containing Rnr1, and why is the activity improved
in the presence of inactive Rnr1? Studies on mouse RNR have shown that
to form a complex with the small subunit, the polypeptide chains of the
large subunit have to dimerize (28). The highly
concentration-dependent catalytic activity shown by Rnr3 in
contrast to Rnr1 (23) and the lower temperature stability of an
Rnr3-containing RNR compared with an Rnr1-containing enzyme indicate
that Rnr3 is less efficient in forming a stable active complex with the
Rnr2/Rnr4 heterodimer. This may be either because Rnr3 does not
readily dimerize or because the Rnr3 dimer has a low affinity to the
Rnr2/Rnr4 heterodimer. An Rnr3/Rnr1 complex makes a more stable RNR
than Rnr3 alone. The sucrose gradient centrifugation experiments
indicate that Rnr3 is less efficient in forming oligomeric structures
than Rnr1, and we believe that this weakness is a major reason for the
low catalytic activity of Rnr3 compared with Rnr1.
Addition of increasing amounts of inactive Rnr1-C428A to an assay
mixture containing Rnr2/Rnr4 heterodimer and Rnr3 or Rnr1, to our
surprise, did not result in inhibition as a consequence of competition
for the small subunit. Such an inhibition is clearly observed when
inactive E. coli R1-C439A protein is added to an assay
mixture containing bacterial R1 and R2 proteins (29). Obviously, the
inactive Rnr1-C428A protein is not very effective in competing with
active Rnr1 dimers for the Rnr2/Rnr4 heterodimer. A difference between
the yeast and bacterial RNR systems is that in yeast, maximally only
50% of the complexes between an Rnr1/Rnr1-C428A dimer and the
Rnr2/Rnr4 heterodimer are active because Rnr4 completely lacks the
catalytically essential tyrosyl radical. The initial stimulation of the
catalytic activity observed when inactive Rnr1-C428A was added to
Rnr1-containing RNR suggests that there is a preferential formation of
enzyme complexes in which active Rnr1 is binding to the catalytically
active, tyrosyl radical-containing Rnr2, while inactive Rnr1-C428A is
binding to the catalytically inactive Rnr4 in the Rnr2/Rnr4
heterodimers. The mechanism for this apparent active
site-dependent orientation is not known. The inability of
the inactive Rnr1-C428A to compete efficiently for the small subunit
may be explained by the same mechanism.
Stimulation of the Rnr3-catalyzed CDP reduction by dATP indicates that
Rnr3 lacks a functional allosteric activity site. This behavior is
similar to that of the mouse R1 D57N protein with a mutation in the
allosteric activity site (15). Therefore, similar to ATP, dATP may only
bind to the allosteric specificity site where both nucleotides induce
pyrimidine nucleotide specificity. The lack of a functional allosteric
activity site may explain the inefficient binding of Rnr3 to the small
subunit because effector binding increases affinity between the large
and small subunits in mouse RNR (28). A low affinity for nucleotide
effector binding to Rnr3 is apparent from the ATP stimulation curve in
Fig. 6A where the Rnr3-catalyzed reduction shows an S-shaped
activity curve with increasing ATP concentrations, and Rnr1 and
Rnr3/Rnr1-C428A show hyperbolic curves. A low Rnr3 affinity for
effector nucleotides also explains the high concentrations of ATP/dATP
required for maximal activity and the rather limited effect of ATP on
the oligomerization of Rnr3 as compared with Rnr1. The unexpected
observation that the allosteric regulation of
Rnr3/Rnr1-C428A-containing RNR is very similar to the allosteric
regulation of Rnr1-containing RNR (but not to Rnr3-containing RNR)
strongly indicates that the allosteric activity site present in the
catalytically inactive Rnr1-C428A cross-talks with the catalytic site
in Rnr3. Such a cross-talk between two polypeptide chains of the large
subunit of RNR has been suggested earlier but never before demonstrated
in direct experiments. Binding of dATP to the allosteric activity site
of Rnr1-C428A must signal to the catalytic site of Rnr3 through
conformational changes in the two polypeptide chains.
We thank Steven Elledge, Xiaolan Zhao, and
Rodney Rothstein for providing yeast strains and Elizabeth Murchison
for helpful comments.
*
This work was supported by the Swedish Research Council and
the Medical Faculty of Umeå University and by fellowships from the
Wenner-Grenska Samfundet and the Royal Swedish Academy of Sciences
(to V. D.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, March 13, 2002, DOI 10.1074/jbc.M201553200
The abbreviations used are:
RNR, ribonucleotide
reductase;
4-NQO, 4-nitroquinoline-1-oxide.
Yeast DNA Damage-inducible Rnr3 Has a Very Low Catalytic Activity
Strongly Stimulated after the Formation of a Cross-talking Rnr1/Rnr3
Complex*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
2 heterotetramer. The
homodimer 2 is referred to as the large subunit, and the
homodimer 2 is referred to as the small subunit. Both
subunits are required for activity. The small subunit contributes a
tyrosyl free radical essential for catalysis. The large subunit
contains one catalytic active site and two allosteric sites on each polypeptide; the allosteric specificity site regulates the balance
among the four dNTP pools, and the allosteric activity site regulates
the total dNTP pool size by monitoring the dATP/ATP ratio (15). When
the dATP pool reaches a certain level, the RNR is down-regulated by dATP feedback inhibition.
'
heterodimer containing Rnr2 and Rnr4 (23). Rnr4, thus far a unique
variant of the small subunit polypeptide, does not form a tyrosyl
radical; its role is to correctly fold and stabilize the
radical-storing Rnr2. A nucleotide sequence comparison of
RNR1 and RNR3 shows 80% identities and 90%
similarities of the coding sequences. However, the expression patterns of RNR1 and RNR3 are completely
different (5). RNR1 is essential for mitotic viability, and
its transcription is cell cycle-regulated with maximal mRNA levels
present during S phase. The RNR3 transcript is nearly absent
during normal growth but appears after DNA damage. Thus, during normal
growth the large subunit is an 2 homodimer containing
only Rnr1. Although the transcriptional regulation of the
RNR3 gene has been studied in great detail, the biochemical
properties and the amount of the Rnr3 protein in a yeast cell after DNA
damage are unknown; also unknown is the ratio between the Rnr1 and Rnr3 proteins.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
rnr1::HIS3)
containing a 2-µ plasmid with the RNR3 gene (Apr TRP1 2 µ GAP-RNR3)
under a strong and constitutive glyceraldehyde 3-phosphate
dehydrogenase promoter. Yeast cells were disrupted in a Bead-Beater
(Biospec Products, Bartlesville, OK), and the rest of the purification
was carried out as described for the bacterially expressed Rnr1. To
construct the Rnr1-C428A, the pET RNR1 expression plasmid (23) was
mutagenized using the QuikChange site-directed mutagenesis kit
(Stratagene) and the oligonucleotide primers 5'-ATC AAG TCA TCA AAC TTA
GCC TGT GAA ATT GTT GAA TAC-3' and 5'-GTA TTC AAC AAT TTC
ACA GGC TAA GTT TGA TGA CTT GAT-3'. The correct sequence
was confirmed by DNA sequence analysis. Expression and purification of
Rnr1-C428A were carried out as described for Rnr1 (24).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (25K):
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Fig. 1.
DNA damage induction of Rnr1 and Rnr3.
Crude extracts (37 µg of protein per lane) of yeast cells grown with
and without treatment by 4-NQO were analyzed for Rnr1 and Rnr3 by
immunoblotting as described under "Experimental Procedures."
A, lane 1, Rnr1 after 4-NQO induction; lane
2, Rnr1 without induction; lane 3, Rnr3 after 4-NQO
induction; lane 4, Rnr3 without induction. The lower
band in lanes 3 and 4 with the anti-Rnr3
antibodies is an unspecific band always observed with these antibodies.
M, molecular mass markers, 110 and 90 kDa. B,
determination of the amounts of Rnr1 in a yeast crude extract after
4-NQO induction (lanes 3, 5, 7,
9, 11, and 13) by comparing with
increasing amounts of pure recombinant Rnr1 (lane 1, 10 ng;
lane 2, 20 ng; lane 4, 40 ng; lane 6,
60 ng; lane 8, 80 ng; lane 10, 120 ng; lane
12, 160 ng, and lane 14, 200 ng). Yeast extract (37 µg) was estimated to contain approximately 100 ng of Rnr1.
C, determination of the amounts of Rnr3 in a yeast crude
extract after 4-NQO induction (lanes 3, 5,
7, 9, 11, and 13) by
comparing with increasing amounts of pure recombinant Rnr3 (lane
1, 5 ng; lane 2, 10 ng; lane 4, 20 ng;
lane 6, 30 ng; lane 8, 40 ng; lane 10,
50 ng; lane 12, 60 ng, and lane 14, 70 ng). Yeast
extract (37 µg) was estimated to contain approximately 10 ng of
Rnr3.
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Fig. 2.
SDS-PAGE analyses of purified recombinant
Rnr3 isolated from yeast and recombinant Rnr1-C428A isolated from
E. coli. Lane 1, molecular mass markers at 205, 116, 97.4, 66, and 45 kDa; lane 2, 6.5 µg of Rnr3; lane
3, 3 µg of Rnr1-C428A.
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Fig. 3.
Catalytic activity of Rnr3 assayed in the
presence of an excess of Rnr2/Rnr4 heterodimer before and after the
addition of a constant amount of Rnr1. Increasing amounts of Rnr3
were assayed for 30 min at 30° C in the presence of 5 mM
ATP and 8.4 µg of Rnr2/Rnr4 heterodimer ( 
). The
experiment was repeated as before, but 0.3 µg of Rnr1 was added to
each reaction mixture (
). The middle curve ( 
)
represents the calculated sum of activities of 0.3 µg of Rnr1 and
increasing amounts of Rnr3 assayed separately.
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Fig. 4.
Catalytic activity of Rnr3 or Rnr1 assayed in
the presence of Rnr2/Rnr4 heterodimer and without or with inactive
Rnr1-C428A. A, Rnr3 (2.6 µg) was assayed for 30 min
at 26° C with 5 mM ATP and 1.1 µg of Rnr2/Rnr4
heterodimer in the presence of increasing amounts of Rnr1-C428A
( ). No activity was detected when Rnr3 was omitted from the
reaction mixtures (). B, Rnr1 (1 µg) was assayed
for 30 min at 30° C with 5 mM ATP and 0.56 µg of
Rnr2/Rnr4 heterodimer in the presence of increasing amounts of
Rnr1-C428A ().
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Fig. 5.
Temperature optima for ribonucleotide
reduction catalyzed by Rnr3, Rnr3/Rnr1-C428A, or Rnr1 in the presence
of Rnr2/Rnr4 heterodimer. Rnr3 (13 µg) was assayed in the
presence of 5 mM ATP and 1.1 µg of Rnr2/Rnr4 heterodimer
for 30 min at increasing temperatures ( - - ). The same
assay was performed for an Rnr3 (13 µg)/Rnr1-C428A (5.4 µg) mixture
(). Results of an assay of Rnr1 (6 µg) with 5 mM
ATP and 1.9 µg of Rnr2/Rnr4 heterodimer for 10 min at increasing
temperatures (
- - ) are shown.
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Fig. 6.
Cross-talk between the allosteric sites in
Rnr1-C428A and the catalytic site in Rnr3. A, reduction
of CDP by Rnr1, Rnr3/Rnr1-C428A, or Rnr3 in the presence of Rnr2/Rnr4
heterodimer and increasing concentrations of ATP. Rnr1 (1.4 µg) and
Rnr2/Rnr4 heterodimer (7 µg) assayed at 30° C, dCDP formed per
minute ( ); Rnr3 (10 µg) assayed at 25° C in the presence
of 2.8 µg of Rnr2/Rnr4 heterodimer, dCDP formed during 30 min
(); and Rnr3 (10 µg)/Rnr1-C428A (38 µg) assayed at 25° C
in the presence of Rnr2/Rnr4 heterodimer (2.8 µg), dCDP formed during
5 min (). In all experiments, magnesium acetate was added in a
2.5 molar excess over ATP. B, inhibition of the
ATP-stimulated CDP reduction by dATP. All reactions were incubated at
25° C, and all mixtures contained 5 mM ATP and 20 mM magnesium acetate. Rnr3 (8.4 µg) and Rnr2/Rnr4
heterodimer (8.4 µg), dCDP formed during 30 min at increasing
concentrations of dATP (); Rnr3 (4.2 µg)/Rnr1-C428A (4.2 µg) in the presence of Rnr2/Rnr4 heterodimer (8.4 µg), dCDP formed
during 30 min at increasing dATP concentrations (); and Rnr1
(0.8 µg) in the presence of Rnr2/Rnr4 heterodimer (8.4 µg), dCDP
formed per minute at increasing dATP concentrations ().
View larger version (21K):
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Fig. 7.
Analyses of the influence of allosteric
effectors on the oligomeric structure of yeast Rnr1 and Rnr3 by sucrose
gradient centrifugation. The ordinate gives protein concentration
in each fraction assayed by the Bradford method. The arrow
indicates the position of catalase. A, Rnr3 (150 µg) was
centrifuged in a 5-20% linear sucrose gradient containing 20 mM HEPES buffer, pH 7.4, 100 mM potassium
acetate, 10 mM magnesium acetate, and 2 mM
dithiothreitol (thin solid line), in the presence of 0.1 mM dTTP (thin dashed line), or in the presence
of 2 mM ATP (thick dashed line). B,
Rnr1 (500 µg) was sedimented in the same buffer as above without
effectors (thin solid line), in the presence of 0.1 mM dTTP (thin dashed line), or in the presence
of 2 mM ATP (thick dashed line).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Cold Spring Harbor
Laboratory, 1 Bungtown Rd., P. O. Box 100, Cold Spring Harbor, NY
11724. Tel.: 516-367-8384; Fax: 516-367-8454; E-mail:
chabes@cshl.org.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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