TIBS 17 - MARCH 1992
References I Hanks, S. K. and Quinn, A. M. (1991) Methods Enzymol. 200, 38-62 2 Wilks, A. F. (1989) Proc. Natl Acad. Sci. USA 86, 1603-1607 3 Hanks, S. K., Quinn, A. M. and Hunter, T. (1988) Science 241, 42-52 4 Featherstone, C. and Russell, P. (1991) Nature 349, 808-811 5 Ben-David, Y. et al. (1991) EMBO J. 10, 317-325 6 Johnson, K. W. and Smith, K. A. (1991) J. Biol. Chem. 266, 3402-3407 7 Howell, B. W. et al. (1991) Mol. Cell. Biol. 11, 568-572 8 Seger, R. et al. (1991) Proc. Natl Acad. Sci. USA
88, 6142-6146 9 Payne, D. M. et al. (1991) EMBO J. 10, 885492 10 Lundgren, K. et al. (1991) Cell 64, 1111-1122 11 Stern, D. F. et al. (1991) Mol. Cell. Biol. 11, 987-1001 12 Icely, P. L. et al. (1991) J. Biol. Chem. 266, 16073-16077 13 Lindberg, R. A., Thompson, D. P. and Hunter, T. (1988) Oncogene 3, 629-633 14 Dailey, D. et al. (1990) Mol. Cell. Biol. 10, 6244-6256 15 Neigeborn, L. and Mitchell, A. P. (1991) Genes Dev. 5, 533-548 16 Shero, J. H. and Hieter, P. (1991) Genes Dev. 5, 549-560
and is catalysed by the enzyme ribonucleoside diphosphate reductase (ribonucleotide reductase, RNR). Ribonucleotide reductase is a highly regulated enzyme and, owing to its central role in the control of DNA synthesis, has been extensively studied in a number of organisms. Among eukaryotes, the mammalian enzyme has been characterized in the greatest detail and its general biochemical features are typical of other eukaryotic, prokaryotic and viral ribonucleotide reductases including those from Saccharomyces cerevisiae, herpes simplex virus (HSV), Escherichia coil and phage T4. We will briefly review the biochemical properties of RNR here, but for more detailed biochemical information, the reader is referred to several recent reviews ],2. RN~ is an enzyme of (z2[~2 structure. The large subunit, R1, has been purified to homogeneity from calf thymus and is a dimer of monomeric molecular mass 90 kDa (Refs 3, 4). Each monomer contains two distinct binding sites for deoxynucleoside triphosphates which act as allosteric regulators of the enzymatic activity. One site controls the substrate specificity of RNR and is S. J. Elledge, Z. Zhou and J. B. Allen are at
the Departmentof Biochemistryand Institute for MolecularGenetics, BaylorCollegeof Medicine, Houston,TX 77030, USA.
407-414 19 Knighton, D. R. et al. (1991) Science 253,
414-420 20 Hanks, S. K. (1991) Curr. Opin. Struct. Biol. 1,
369-383 21 Parker, L. L. et al. (1991) EMBO J. 10,
1255-1263 22 Krek, W. and Nigg, E. A. (1991) EMBO J. 10,
3331-3341 23 Ahn, N. G. et al. (1991) J. Biol. Chem. 266,
4220-4227 24 Gomez, N. and Cohen, P. (1991) Nature 353,
Ribonucleotide reductase: regulation, regulation, regulation
DEOXYRIBONUCLEOTIDES, the precursors for DNA synthesis, are produced by direct reduction of the corresponding ribonucleotides in all organisms thus far examined. With the exception of lactobacilli, the reaction is of the form: NDP + reductant-(SH)2 dNDP + reductant-(S-S)
17 Tan, J. L. and Spudich, J. A. (1990) Mol. Cell. Biol. 10, 3578-3583 18 Knighton, D. R. et al. (1991) Science 253,
Ribonucleotide reductase (RNR) catalyses the rate limiting step in the production of deoxyribonucleotides needed for DNA synthesis. It is composed of two dissimilar subunits, R1, the large subunit containing the allosteric regulatory sites, and R2, the small subunit containing a binuclear iron center and a tyrosyl free radical. Recent isolation of the mammalian and yeast RNR genes has shown that, in addition to the well documented allosteric regulation, the synthesis of the enzyme is also tightly regulated at the level of transcription. The mRNAs for both subunits are cell-cycle regulated and, in yeast, inducible by DNA damage. Yeast encode a second large subunit gene, RNR3, that is expressed only in the presence of DNA damage. This regulation is thought to provide a metabolic state that facilitates DNA replicational repair processes.
responsible for maintaining balanced nucleotide pools, thus facilitating optimal fidelity of DNA replication (Fig. 1). The second site monitors the ATP/dATP ratio and modulates the overall activity of the enzyme, presumably to ensure that sufficient dNTPs are produced for DNA synthesis without depleting ribonucleotides needed for RNA synthesis 3. The smaller subunit, R2, is a dimer of monomeric molecular weight 45 kDas. Each monomer contains a binuclear ferric iron center and a tyrosyl free radical essential for activity6,8. It was first purified to homogeneity from hydroxyurea-resistant, R2 overproducing mouse cells 7 and recently pure recombinant mouse protein has been obtained in large scale from E. coil s. The chemotherapeutic agent hydroxyurea is a potent inhibitor of RNR activity that functions by quenching the
© 1992, Elsevier Science Publishers, (UK) 0376-5067/92/$05.00
free radical on R2. Reactivation of RNR requires molecular oxygen6,9. Since the large subunit, R1, contains sites for allosteric regulation and the small subunit, R2, contains residues involved in catalysis, these subunits have often been referred to as the regulatory and catalytic subunits, respectively. However convenient, these descriptions are not completely accurate because neither protein alone has catalytic activity. Furthermore, although RI has regulatory functions, it is not solely regulatory, since it binds nucleoside diphosphate substrates and contains redox active sulfhydryls12. Genes encoding ribonucleotide reductase in
5. cerevisiae The properties of the three genes that encode ribonucleotide reductase in
TIBS 17 - MARCH 1 9 9 2
Only one yeast gene for the small subunit, R2, has been identified, RNR2u,~2. RNR2 encodes a protein of 46 kDa that shares 60% amino acid identity with the mammalian homolog (mouse). That RNR2 is essential for mitotic viability has been shown by genetic disruption experiments using both tetrad analysis and a plasmid sectoring assay. Mutations that reduce RNR2 function but are not lethal show hypersensitivity to hydroxyurea and to killing by methyl methanesulfonate 0VIMS)11. Since MMS damages DNA, hypersensitivity to this agent demonstrates a role for ribonucleotide reductase in DNA repair processes. Spores bearing an rnrl or rnr2 null mutation show a cdc terminal phenotype of large budded cells upon germination, characteristic of mutations in genes required for DNA replication 1°,]~.
(a) Substrate Specificity (ATP, dATP, dTTP, dGTP)
Activity (ATP, dATP)
Cell cycle regulation
POC O , OH
R-S 2 + H 2 0
Rguro 1 (a) Model of eukaryotic ribonucleotide reductase. The R1 subunit contains two classes of allosteric sites, regulating the substrate specificity or overall activity. The R2 subunit contains the antiferromagnetically coupled iron center and the tyrosyl free radical. The active site is constructed from parts of the R1 subunit contributing redox active sulfhydryls and parts of the R2 subunit contributing the free radical. (b) Reaction catalysed by ribonucleotide reductase.
S. cerevisiae are summarized in Table I. Two genes, RNRI and RNR3, encode the large subunit R1~°. They are 80% identical in the portions of their genes that have both been sequenced (~150 of 790 amino acids), and 60% identical to their mammalian homolog. RNRI is an essential gene, while deletion of RNR3 has no obvious phenotype. However," RNR3 encodes an active protein since RNR3 cloned on the high copy number 2 pm plasmid is capable of complementing a null allele of RNRP °. Northern analysis
of RNA extracted from logarithmically growing cells showed that RNRI encodes a 3.4 kb mRNA, but the RNR3 mRNA could not be detected. Table I. Properties of the S. cerevisiae RNR genes Gene
The eukaryotic cell cycle is a cascade of tightly regulated events that culminate in the duplication of a ceil. Many complex macromolecular structures must be assembled and disassembled with striking temporal and spatial precision. This degree of complexity necessitates the existence of a sophisticated regulatory network capable not only of coordinating these events, but also of correcting mistakes that occur during these complex processes. One type of regulation is restriction of certain cell cycle functions to particular periods of the cell cycle when they are needed. For example, the restriction of DNA replication to a defined period of the cell cycle, S phase, is accomplished, at least in part, through the temporal modulation of the activity and expression of gene products needed specifically in S phase. In S. cerevisiae not only are genes encoding the enzymatic machinery for DNA synthesis cell-cycle regulated, but so are several of the enzymatic activities involved in the production of the dNTP precursors needed for DNA synthesis (see below). The significance of the cell-cycle regulation with respect to the overall capacity to synthesize DNA outside of S phase has not been determined. However, the effect on cells that experience prolonged periods outside of S phase, such as those in stationary phase, may be more pronounced with respect to the absence of S-phase-specific proteins reducing the capacity to synthesize DNA. Ribonucleotide reduction in yeast was investigated by Lowden and Vitols ~s who
TIBS 17 - M A R C H 1 9 9 2
observed maximum activity in S phase. Cell cycle regulation of the RNR genes was investigated by Elledge and Davis ~°, who observed that there was a greater than tenfold fluctuation in RNR1 mRNA concentrations during the cell cycle, while the RNR2 transcript showed a modest twofold fluctuation in cells moving synchronously through the cell cycle (Table II). Thus, the cell cycle fluctuation of ribonucleotide reductase activity is likely to be primarily due to modulation of the RNRI transcript although protein concentrations must be measured to confirm this point. In mammalian cells, the amount of R1 protein is constant and that of R2 fluctuates. However, mRNA of both genes are cell-cycle regulated 14. Transcription of RNR1 is coordinately regulated with transcription of the POLl gene that encodes DNA polymerase 1~°,~s(Fig. 2). Both are induced in late G1 phase, directly preceding the initiation of DNA replication. This coordination makes biological sense since both enzymes are required at precisely the same time in the cell cycle, at the
Table II. Summary of RNR cell cycle and DNA damage regulation Gene
DNAdamage Cellcycle inducibility regulation M/ulrepeats
Sequences that mediate the cell cycle control of RNR
aDNA fragments from the RNR1 promoter can confer up to 30-fold induction upon the heterologous cyc4acZ reportergene, indicatingthat it shares the same capacityto sense damage as RNR2. b'lhe putativeelementin RNR2is a near match, AGCGCG,to the M/u1sequence.
Sequence comparison between the RNRI and RNR3 promoter regions identified a duodecamer repeat of sequence [A/T][A/T][A/T]AGCGCT[A/T][A/T][A/T] that is repeated twice in RNR3 and four times in the RNRI promoter (Elledge, unpublished data, Table II). These repeats contain at their core the recognition site for the restriction enzyme
Mlul (for an excellent review see Ref.
start of S phase. Further analysis of the cell cycle regulation of RNRI and POLl revealed that the ability to induce the RNRI and POLl transcripts after arrest in G1 is dependent upon protein synthesis ~°. In this experiment, cells are synchronized in G1 by addition of the mating factor ~ and allowed to re-enter the cell cycle in the presence or absence of cycloheximide. This protein synthesis dependence is probably due to the need to synthesize the G1 cyclins
Rgum 2 A summary of the biochemical and genetic events between Start and the induction of S-phase-specificgene expression and the induction of gene expression by DNA damage. The names of genes involved are in capital letters. Only those genes known to have a regulatory role are listed. Other genes that operate at Start are presumed to have a role but are not listed because their role in the induction of S phase genes has not yet been tested. A negative feedback loop from G2 to S-phase-expression is hypothesized because transcription is reduced after S phase. However, the loop is not meant to suggest any particular mechanism for turn off. The DUN and CRTgenes are lacking gene numbers because it is not yet known which genes act at what points in the pathway. Furthermore, assignment of functional order is arbitrary. TF stands for tran-
that are repressed upon addition of tz factor and which are necessary for passage through Start.
16). This repeat has been found in a number of S-phase-regulated promoters including POLl (DNA polymerase 1) 17, POL3 (DNA polymerase III), PRI1 and PRI2 (DNA primase subunits) 16, CDC9 (DNA ligase) ~8, CDC8 (thymidylate kinase) TM, CDC21 (thymidylate synthase) ~9, and CDC6 which is needed for DNA synthesis 16. The RNR2 promoter contains one near match to the consensus sequence AGCGCG. Mutations in the CDC21 promoter which destroy
T I [ 1 G1/S phase
Protein synthesis (cyclins ?)
represents a hypothetical DNA damage signal that may or
may not be shared among the different stimuli. The arrow leading to cell-cycle arrest is arbitrarily placed leading directly from hydroxyureaor DNA damage, but may be completely or partially overlapping with the pathways leading to increased transcription.
TIBS 17 -
these two repeats abolish cell-cycle regulation2°.2L Oligonucleotides representing the core of the consensus sequence were synthesized and shown to act as a cell-cycle regulated upstream activating sequence (UAS) when present in multiple copies in front of the CYC1 promotet 2t,22. A DNA binding protein that binds these sequences has been detected in crude extracts and the binding pattern shows a cell-cycle periodicity=. However, this pattern fails to mimic completely the expression of the S-phase specific genes. More recently, a 17 kDa protein has been purified that binds to the MluI repeats, but whether it is the same protein that shows cellcycle regulated binding properties is not known 23. It should be noted that, although it has two copies of the MluI repeat sequence, it is not yet known whether transcription from RNR3 is cell-cycle regulated when it is expressed.
urea treatment, there is evidence in mammalian cells that RNR synthesis is also modulated post-transcriptionally28. Induction of RNR2 by DNA damage is independent of protein synthesis and cell cycle stage, and sequences from the RNR2 regulatory region are able to confer DNA-damage inducibility upon heterologous promoters 24,29. Insensitivity to inhibition by cycloheximide formally eliminates the de novo synthesis of a positive activator as a model for DNA damage induction, and leaves two equally plausible molecular models: (1) post-translational modification of a positive activator; or (2) inactivation of a negatively acting factor. Because so many genes involved in DNA synthesis are inducible by DNA damage, perhaps this response should be viewed as a partial reactivation of S phase without origin initiation.
detected. Relatively minor selective advantages have a much more pronounced effect in competitive growth experiments carried out over extended periods of time.
Induction signals: nucleotide depletion or DNA damage?
What are the signals to which the RNR promoters respond? Clearly nucleotide depletion can induce RNR2 because treatment with methotrexate or hydroxyurea can induce high levels of synthesis. It is sound biochemical logic that an enzyme whose role is to synthesize deoxyribonucleotides would be induced in response to their depletion. It is also possible that the DNA damage induction proceeds through nucleotide depletion brought about by repair synthesis. Genetic experiments involving the rad4-2 mutation, which blocks the incision step of excision repair, and 4-NQO, a DNA damaging Why two regulatory subunit genes? Under normal vegetative conditions, agent whose lesions are repaired by the only RNR1 and RNR2 are expressed, excision repair pathway, show that DNA damage-inducibilityof RNR, a reactivation of S-phase? and thus the ribonucleotide reductase blocking excision repair actually enDue to the temporal compartmental- in the cell presumably contains two hances inducibility and strongly sugization of the cell cycle, much of the copies of each subunit ((~12132). gests that the regulators of RNR2 accapacity to synthesize DNA is restricted However, in the presence of DNA dam- tually respond to the levels of DNA to S phase. However, circumstances age, the second large subunit gene is damage present 24. Whether nucleotide can arise when this capacity is needed induced, producing at least one ((z22J32) depletion and DNA damage signals are outside of the period in which it is nor- and possibly two, ((~1(~2132) additional identical and are mediated via the same mally present. For example, repair of forms of ribonucleotide reductase. proteins remains to be determined. certain types of DNA damage requires Yeast strains mutant for RNR3 have no the ability to synthesize DNA. If repair obvious growth defects, and are not Sequences involvedin the DNA damage is to proceed optimally outside of S sensitive to hydroxyurea or DNA dam- response Detailed deletion analysis of the phase, cells must possess the capacity aging agents (Elledge, unpublished to synthesize DNA in other phases of data). What then is the role of RNR3?. It RNR2 regulatory region has implicated the cell cycle. In fact, several genes with must confer some selective advantage both a positive and a negative regucell-cycle regulated activities involved for the cell to have conserved both its latory element in this response and in DNA synthesis are inducible by DNA function and tight regulation. One possi- identified a 70 bp fragment called the damage (POLP 2, CDC914,CDC87), as are bility is that the RNR3 protein has DRE (DNA damage responsive element) all three genes encoding ribonucleo- altered regulatory properties allowing that can confer some DNA-damage tide reductase (RNRI, RNR2, and the cell to survive certain types of inducibility upon a heterologous proRNR31°-12'24'2s). The induction of these stress that we have yet to duplicate in moter 25,29.Originally there were thought genes in response to the stress of DNA the laboratory. For example, biological to be four proteins that bound this eldamage is thought to produce a fungicides are rampant in the wild, and ement, RRF1, 2, 3 (RNR regulatory facmetabolic state that facilitates DNA are often targeted to inhibit key regu- tors) and the RAP1 (GRF1) protein, an replicational repair processes. RNRI, latory enzymes. Perhaps the evolution- abundant protein that can act to actiRNR2, and RNR3 are all inducible at the ary history of S. cerevisiae included vate or repress gene expression delevel of transcript accumulation by growth in an ecosystem in which pending upon context 3°,31.Recent experagents that block DNA synthesis, such inhibitors of ribonucleotide reductase iments by Zhou and Elledge (unpubas hydroxyurea and methotrexate, or were a commonly employed competi- lished data) have shown that the seby agents that damage DNA, such as UV tive strategy. Duplication of the target quence to which RRF1 binds is actually a light, MMS, and 4-NQO (4-nitroquino- gene could facilitate the rapid evolution weak RAPI binding site, and that RRF1 line-l-oxide). RNRI is inducible three- to of drug-resistant variants. Alternatively, is RAP1, suggesting that there are two fivefold, RNR2 is inducible 2~fold, and the RNR3 gene may play a role in a non- RAP1 binding sites in this promoter. A RNR3 is inducible 100- to 500-fold vegetative function of yeast such as repressing sequence has also been (Table II). RNR3 was found to be ident- meiosis. RNR3 may play a marginal role detected in the region conferring DNA ical to the previously isolated gene in cell survival under certain conditions damage inducibility, but it is clearly DIN126,27, isolated on the basis of its that are not immediately obvious in not sufficient to mediate the response DNA damage inducibility. In addition to short-term laboratory experiments alone. The presence of DNA damage transcriptional responses to hydroxy- where only severe defects can be does not alter the pattern of DNA bindl22
TIBS 1 7 - M A R C H
ing proteins detected, nor their abundance or affinity. Furthermore, no factors common to any two of the RNR promoters have been detected. Detailed analysis of the deletion studies on RNR2 suggest that more than one element may be capable of transducing the DNA damage signaF5,29. The DRE element can confer DNA damage inducibility upon a lacZ reporter gene. Deletions from the 5' end which remove this sequence (and all upstream sequences) destroy DNA damage inducibility25. However, deletions which remove most of this element but retain sequences 5' to this element in the native promoter, retain some DNA damage-inducibility suggesting that there are sequences upstream of the DRE that are capable of transducing the DNA damage signal 29. This issue may be further complicated by the fact that RNR2 shows weak cell-cycle regulation and that most DNA damaging treatments elongate S phase giving rise to artificial induction due to synchronization.
Mutationsthat alter the DNAdamage sensorypathway T w o t y p e s of m u t a t i o n s h a v e b e e n
identified (Zhou and Elledge, in preparation) that alter the regulation of the RNR3 gene. The crt mutants (constitutive RNR3 transcription) show high levels of RNR3 expression in the absence of DNA damage. These mutations are likely to be in genes that negatively regulate RNR3 or in genes that in their mutant form produce an endogenous DNA damage signal, dun mutants (DNA damage uninducible) fail to induce RNR3 in response to DNA damage or hydroxyurea treatment. The dun genes are more
likely to be sensors and transducers of the damage signals. It will be interesting to determine whether the pathway that regulates the transcriptional response to DNA damage and hydroxyurea overlaps with the cell cycle arrest in response to DNA damage, the rad9 pathway32, or in response to hydroxyurea treatment, a rad9 independent pathway (Ref. 10; T. Weinert, pers. commun.). Little is currently known about the genes identified by the crt and dun mutations. However, they are likely to be mediators of the DNA damage response because some affect the regulation of RNR1 and RNR2 as well, and their analysis will be critical to the molecular elucidation of this regulatory pathway.
Acknowledgements We thank M. Kuroda, R. Lin and K. Becherer for critical comments on the manuscript and are indebted to two anonymous reviewers for their excellent suggestions. This work was supported by the grants NIGMS1R01GM44664 and Ql186 from the Robert A. Welch Foundation to S. J. E. Z. Z. is a Robert A. Welch Predoctoral Fellow and S. J. E. is a P. E. W. Scholar in the Biomedical Sciences.
References 1 Stubbe, J. (1990) J. Biol. Chem. 256, 5329-5332 2 Reichard, P. (1988) Annu. Rev. Biochem. 57, 349-374 3 Thelander, L., Erikson, L. and Akerman, M. (1980) J. Biol. Chem. 255, 7426-7432 4 Caras, I. W. et al. (1985) J. Biol. Chem. 260, 7015-7022 5 Thelander, L. and Berg, P. (1986) Mol. Cell. Biol. 6, 3433-3442 6 Petersson, L. et al. (1980) J. Biol. Chem. 255, 6706-6712 7 Thelander, M., Grasiund, A. and Thelander, L.
(1985) J. Biol. Chem. 260, 2737-2741 8 Mann, G. J. et al. (1991) Biochemistry30, 1939-1947 9 Harder, J. and Follmann, H. (1990) Free Rad. Res. Comms. 10, 281-286 10 Elledge, S. J. and Davis, R. W. (1990) Genes Dev. 4, 740-751 11 Elledge, S. J. and Davis, R. W. (1987) Mol. Cell. Biol. 7, 2783-2793 12 Hurd, H. K., Roberts, C. W. and Roberts. J. W. (1987) Mol. Cell. Biol. 7, 3673-3677 13 Lowden, M. and Vitols, E. (1973) Arch. Biochem. Biophys. 158, 177-184 14 Bjorklund, S., Skog, S., Tribukait, T. and Thelander, L. (1990) Biochemistry 29, 5452-5458 15 Johnston; L. H. et al. (1987) Nucleic Acids Res. 15, 5017-5030 16 Andrews, B. J. and Herskowitz, I. (1989) J. Biol. Chem. 265, 14057-14060 17 Barker, D. G., White, J. M. and Johnston, L. H. (1985) Nucleic Acids Res. 13, 8323-8337 18 White, J. et al. (1988) Exp. Cell Res. 171, 223-231 19 Storms, R. K. et al. (1984) Mol. Cell. Biol. 4, 2858-2864 20 Mclntosh, E. M., Ord, R. W. and Storms, R. K. (1988) Mol. Cell. Biol. 8, 4616-4624 21 Mclntosh, E. M., Atkinson, T., Storms, R. K. and Smith, M. (1991) Mol. Cell. Biol. 11, 329-337 22 Lowndes, N. F., Johnson, A. L. and Johnston, L. H. (1991) Nature 350, 247-250 23 Verma, R., Patapoutian, A., Gordon, C. B. and Campbell, J. L. (1991) Proc. Natl Acad. Sci. USA 88, 7155-7159 24 EIledge, S. J. and Davis, R. W. (1989) Mol. Cell. Biol. 9, 4932-4940 25 Elledge, S. J. and Davis, R. W. (1989) Mol. Cell. Biol. 9, 5373-5386 26 Ruby, S. W. and Szostak, J. W. (1985) Mol. Cell. Biol. 5, 75-84 27 Yagle, K. and McEntee, K. (1990) Mol. Cell. Biol. 10, 5553-5557 28 McClarty, G. A. et al. (1988) Biochemistry 27, 7524-7531 29 Hurd, H. and Roberts, J. (1989) Mol. Cell. Biol. 9, 5366-5372 30 Buchman, A. W., Kimmerly, W. J., Rine, J. and Kornberg, R. D. (1988)/Viol. Cell Biol. 8, 210-225 31 Shore, D. and Nasmyth, K. (1987) Cell 51, 721-732 32 Weinert, T. A. and Hartwell, L. H. (1988) Science 241, 317-322
get t,e complete
take out a ~ o n a i s ~ ~g in bhis issue, if someone else has ~ \
to one of
CrownHo~e,Linton~ , ~ng, E ~ USA.k 123