dCMP Deaminase Activity Research Articles

Dr Jekyll and Mr Hyde: a strange case of 5-ethynyl-2'-deoxyuridine and 5-ethynyl-2'-deoxycytidine.

5-Ethynyl-2′-deoxyuridine (EdU) and 5-ethynyl-2′-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a replicational marker more frequently than EdC, its cytotoxicity is commonly much higher than the toxicity of EdC. To reveal the reason of the lower cytotoxicity of EdC, we performed a DNA analysis of five EdC-treated human cell lines. Surprisingly, not a single one of the tested cell lines contained a detectable amount of EdC in their DNA. Instead, the DNA of all the cell lines contained EdU. The content of incorporated EdU differed in particular cells and EdC-related cytotoxicity was directly proportional to the content of EdU. The results of experiments with the targeted inhibition of the cytidine deaminase (CDD) and dCMP deaminase activities indicated that the dominant role in the conversion pathway of EdC to EdUTP is played by CDD in HeLa cells. Our results also showed that the deamination itself was not able to effectively prevent the conversion of EdC to EdCTP, the conversion of EdC to EdCTP occurs with much lesser effectivity than the conversion of EdU to EdUTP and the EdCTP is not effectively recognized by the replication complex as a substrate for the synthesis of nuclear DNA.

Isolation and Characterization of a Murine P388 Leukemia Line Resistant to Thiarabine

A murine P388 leukemia line fully resistant to thiarabine was obtained after five courses of intraperitoneal treatment (daily for nine consecutive days). The subline was sensitive as was the parental P388/0 line to 5-fluorouracil, gemcitabine, cyclophosphamide, cisplatin, melphalan, BCNU, mitomycin C, doxorubicin, mitoxantrone, etoposide, irinotecan, vincristine, and paclitaxel, but was cross resistant (at least marginally) to three antimetabolites: palmO-ara-C, fludarabine phosphate, and methotrexate. The deoxycytidine kinase activity in the subline was comparable to that for P388/0, whereas the dCMP deaminase activity was 43% of that for P388/0. No deoxycytidine deaminase activity was detected in either of the leukemias. There appeared to be little, if any, difference in the metabolism of deoxycytidine, cytidine, or thiarabine in the two leukemias.

Chloroviruses Encode a Bifunctional dCMP-dCTP Deaminase That Produces Two Key Intermediates in dTTP Formation

The chlorovirus PBCV-1, like many large double-stranded DNA-containing viruses, contains several genes that encode putative proteins involved in nucleotide biosynthesis. This report describes the characterization of the PBCV-1 dCMP deaminase, which produces dUMP, a key intermediate in the synthesis of dTTP. As predicted, the recombinant protein has dCMP deaminase activity that is activated by dCTP and inhibited by dTTP. Unexpectedly, however, the viral enzyme also has dCTP deaminase activity, producing dUTP. Typically, these two reactions are catalyzed by proteins in separate enzyme classes; to our knowledge, this is the first example of a protein having both deaminase activities. Kinetic experiments established that (i) the PBCV-1 enzyme has a higher affinity for dCTP than for dCMP, (ii) dCTP serves as a positive heterotropic effector for the dCMP deaminase activity and a positive homotropic effector for the dCTP deaminase activity, and (iii) the enzymatic efficiency of the dCMP deaminase activity is about four times higher than that of the dCTP deaminase activity. Inhibitor studies suggest that the same active site is involved in both dCMP and dCTP deaminations. The discovery that the PBCV-1 dCMP deaminase has two activities, together with a previous report that the virus also encodes a functional dUTP triphosphatase (Y. Zhang, H. Moriyama, K. Homma, and J. L. Van Etten, J. Virol. 79:9945-9953, 2005), means that PBCV-1 is the first virus to encode enzymes involved in all three known pathways to form dUMP.

Human cytomegalovirus requires cellular deoxycytidylate deaminase for replication in quiescent cells.

We have previously observed that the expression of two thymidylate biosynthesis enzymes, dihydrofolate reductase and thymidylate synthase (TS), is upregulated in quiescent human fibroblasts infected with human cytomegalovirus (HCMV). Here, we have demonstrated that HCMV increases expression of the cellular deoxycytidylate deaminase (dCMP deaminase), which provides the substrate for TS by converting dCMP to dUMP. We observed an increase in dCMP deaminase protein levels, whereas deoxyuridine triphosphatase (dUTPase), another cellular enzyme that may provide dUMP by hydrolysing dUTP, was undetectable. The essential requirement of cellular dCMP deaminase for productive HCMV replication was further emphasized by showing that a precursor of a potent dCMP deaminase inhibitor, zebularine, suppressed virus replication and DNA synthesis. These results suggest that HCMV exploits the host's dCMP deaminase activity to replicate in quiescent cells.

Gene silencing and endogenous DNA methylation in mammalian cells

It is known that transformed mammalian cells can spontaneously inactivate genes at low frequency by the de novo methylation of promoter sequences. It is usually assumed that this is due to DNA methyl transferase activity, but an alternative possibiity is that 5-methyldCTP is present in these cells and can be directly incorporated into DNA. The ongoing repair of DNA containing 5-methylcytosine will produce 5-methyldeoxycytidine monophosphate (5-methyldCMP), so the question arises whether this can be phosphorylated to 5-methyldCTP. We have tested this using three strains of CHO cells with different levels of 5-methyldCMP deaminase activity. That with the lowest enzyme activity, designated HAM −, has previously been shown to incorporate tritium labelled 5-methyldeoxycytidine into 5-methylcytosine in DNA, with a greater amount of label in thymine. This strain is phenotypically unstable producing cells resistant to bromodeoxyuridine (BrdU) and 6-thioguanine (6-TG) at high frequency. In contrast, the strain with the highest 5-methyldCMP deaminease, designated HAM +, is extremely stable, and the starting strain K1 HAM s1 is intermediate between the HAM − and HAM + phenotypes. We have also shown that human diploid fibroblast strain MRC-5 has a phenotype like HAM +, whereas its SV40 transformed derivative, MRC-5V2 resembles HAM − in having low 5-methyl dCMP deaminase activity, and is phenotypically unstable with regard to 6-TG resistance. It seems that 5-methyldCMP deaminase can be down-regulated in transformed cells, and this can promote de novo methylation by incorporation of 5-methyldCTP derived from 5-methyldCMP.

Similar changes were induced by Cladribine and by gemcitabine, in the deoxypyrimidine salvage, during short-term treatments.

Short term treatments (1-2 hrs) of human tonsillar lymphocytes by Cladribine (2-Chloro-deoxyadenosine, CdA) have suggested a new target for CdA, the inhibition of dCMP deaminase (Sasvári et al. 1994; BBRC 203, 1378). Further investigations have shown, that the dCMP-deaminase activity could be inhibited by 2-Cl-dAMP in cell free extracts of lymphocytes. The pool size of dUMP (measured by an antibody against dUMP) was also decreased in WiDr colon cancer cells by CdA. The new antimetabolite against solid tumours, Gemcitabine (2',2'-difluoro-deoxycytidine, dFdC), had similar effects on the salvage of thymidine (dThd) and deoxycytidine (dCyd) as CdA. The Ki values for 3H-dThd and 3H-dCyd incorporation into DNA were 0.16 uM and 1.0 uM dFdC, respectively. The labeling of the TTP pool increased 6-7 times, while of dCTP pool only 1.5-1.7 times, suggesting a decrease of the size of corresponding pools. Similarly to CdA, the labeling as well as the concentration of dUMP was also decreased by dFdC. Both analogues are able to increase the deoxycytidine kinase activity, necessary for their phosphorylation and therapeutic action in cells. The target(s) for the two different drugs seems to be common.

Regulation of deoxycytidine kinase by deoxycytidine and deoxycytidine 5' triphosphate in whole leukemia and tumor cells.

The clinical importance of the dCTP salvage pathway is given by the fact that deoxycytidine (dCyd) analogs like cytosine arabinoside (ara-C) are activated via the salvage pathway. The non-toxic prodrug ara-C gains its cytotoxic potential by phosphorylation to the 5′-triphosphate ara-CTP. Not only cellular accumulation, but also retention of ara-CTP are known determinants of response to antileukemic treatment. Deoxycytdine kinase is the rate-limiting enzyme for intracellular ara-CTP formation. Experiments performed with purified dCK (dCK) demonstrated that dCTP, the final product of phosphorylation, acts as a potent feed-back inhibitor of dCyd kinase.1,2 In fact, depletion of intracellular dCTP as induced by inhibition of the de novo pathway increases of ara-C phosphorylation.3 Therefore it was concluded that the release of dCTP-mediated feed-back inhibition was responsible for enhanced dCK activity. The present study investigates the role of dCTP for regulation of dCK activity in the cell lines CCRF-CEM (T-cell line), Raji (B-cell line), HL-60 (promyelocytic), and CHO (Chinese hamster ovary). Compared to T-cells, B-cells are characterised by a fast turnover of dNTP and a short retention of ara-CTP. CHO cells were included because of their deficiency in deoxycytidylate (dCMP) deaminase activity. Cellular accumulation of ara-CTP was evaluated as a measure of salvage pathway activity. Inhibition of ribonucleotide reductase as induced by hydroxyurea may cause an activation of the salvage pathway to maintain adequate dCTP levels for DNA synthesis. Cells with a fast dCTP turnover and a greater dependence on the de novo pathway should respond with a greater enhancement of the salvage pathway than cells with long retention of deoxynucleotides and a correspondingly lower dependence on the de novo pathway.4 KeywordsChinese Hamster Ovary CellRibonucleotide ReductaseRaji CellSalvage PathwayDeoxycytidine KinaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Deoxycytidine kinase can be also potentiated by the G-protein activator NaF in cells.

Recently, it has been shown, that 2-Chloro-deoxyadenosine (1), a series of analogues, and other DNA synthesis inhibitors, increased the deoxycytidine kinase (dCK) enzyme activity in different cells, without influencing thymidine kinase isoenzymes (TK1, TK2), dCMP-deaminase and thymidylate synthase (TS) activities (2,3). The dCK activity was 2-4 times higher in analogue treated cells, than in controls, which can not be explained by metabolic pool imbalance induced by the drugs. New mRNA and protein synthesis of dCK could not be detected, thus post-translational modification has been suggested for potentiation the activity of the dCK (1). Because secondary modifications of enzymes usually involve the signalling processes in cells, the universal G-protein activator fluorine ions were tested. dCK activity of human lymph node lymphocytes were increased 2-times, if cells were incubated in the presence of NaF for 1-2 hrs in cultures, while TK activity was not changed. The formation of dUTP from dCyd, was also enhanced by NaF, in parallel of dCK potentiation.

Acyclovir Treatment Stimulates dCMP Deaminase Activity in HSV-1-Infected Cells

Herpes simplex virus (HSV) infection induces enzymes involved in DNA precursor metabolism and DNA synthesis. The level of the DNA precursors dCTP, dTTP and dGTP increases after HSV infection, while the dATP level decreases. Inhibition of viral DNA synthesis by acyclovir (ACV) increases all four deoxyribonucleoside triphosphate levels, in particular that of dTTP. In the present work the dynamics of metabolism of pyrimidine deoxyribonucleotides and changes in the dTTP and dCTP pools in HSV-1-infected HL cells and in infected cells where viral DNA synthesis was inhibited by ACV were studied. In the absence of the drug the major part of the dCDP synthesized was incorporated into DNA. ACV treatment inhibited DNA synthesis completely but increased de novo dTMP synthesis. A continued ribonucleotide reductase activity in ACV-treated HSV-1-infected cells contributes to the increasing dTTP pool. The allosteric regulation of dCMP deaminase activity in HSV-1-infected cells was investigated. The great increase of de novo dTMP synthesis was shown to be due to stimulation of dCMP deaminase activity by an increasing dCTP pool. The high dTTP pool is suggested to be of secondary importance to the dCTP pool in the regulation of dCMP deaminase activity in HSV-1-infected cells.

Enzymic activities of carbohydrate, purine, and pyrimidine metabolism in the Anaeroplasmataceae (class Mollicutes).

Cell-free extracts of two strictly anaerobic mollicutes, Anaeroplasma intermedium 5LA and Asteroleplasma anaerobium 161T, were tested for enzymic activities of intracellular carbohydrate metabolism. Asteroleplasma anaerobium was also tested for enzymes of purine and pyrimidine metabolism. Both organisms had enzymic activities associated with the nonoxidative portion of the pentose phosphate pathway, and with the Embden-Meyerhoff-Parnas pathway. The 6-phosphofructokinase (PFK) of Asteroleplasma anaerobium was ATP-dependent, whereas the PFK of Anaeroplasma intermedium was PPi-dependent. The two anaerobic mollicutes also differed with respect to the enzymes that converted phosphoenolpyruvate (PEP) to pyruvate; Anaeroplasma intermedium had pyruvate kinase activity, but Asteroleplasma anaerobium had pyruvate, orthophosphate dikinase activity (PPi-dependent). Both organisms had lactate dehydrogenase activity which was activated by fructose 1,6-bisphosphate (Fru-1,6-P2). Anaeroplasma intermedium had activity for PEP carboxykinase (activated by Fru-1,6-P2), but Asteroleplasma anaerobium did not. PEP carboxytransphosphorylase activity was not detected in either organism. Anaeroplasma intermedium had malate dehydrogenase and isocitrate dehydrogenase activities, but it had no activities for the three other tricarboxylic acid cycle enzymes examined; Asteroleplasma anaerobium had malate dehydrogenase activity only. Asteroleplasma anaerobium had enzymic activities for the interconversion of purine nucleobases, (deoxy)ribonucleosides, and (deoxy)ribomononucleotides, including PPi-dependent nucleoside kinase, reported heretofore only in some other mollicutes. Asteroleplasma anaerobium could synthesize dTDP by the thymine salvage pathway if deoxyribose 1-phosphate was provided, and it had dUTPase, ATPase, and dCMP kinase activities. It lacked (deoxy)cytidine deaminase, dCMP deaminase, and deoxycytidine kinase activities.

Deoxycytidylate deaminase activity in non-stimulated and phytohemagglutinin-stimulated human lymphocytes, and in leukemic cells.

Deoxycytidylate deaminase isolated from normal human lymphocytes and from mononuclear leucocytes from patients with acute lymphoblastic leukemia, chronic lymphocytic leukemia and acute monocytic leukemia has been characterized in regard to the substrate, dAMP and the allosteric regulators dCTP and dTTP. The enzymes exhibited sigmoidal initial velocity versus dCMP concentration whereas in the presence of the activator, dCTP, Michaelis-Menten kinetics were obtained. At saturating substrate concentrations dTTP acted as an allosteric inhibitor of the enzyme isolated from non-stimulated as well as from stimulated lymphocytes. However, the enzymes isolated from the leukemic cells had lost the allosteric regulation by dTTP. At low substrate concentrations the competitive inhibitor, dAMP, activated all the enzymes. This activation was abolished in the presence of dCTP which indicates that dAMP might be involved in the regulation of dCMP deaminase activity and thus influence the dCTP and dTTP pools under physiological conditions.

Sequence and Expression of the dCMP Deaminase Gene (DCD1) of Saccharomyces cerevisiae

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae.

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

Effect of the dialdehyde derivative of 5′-deoxyinosine on pyrimidine deoxyribonucleoside metabolism in L1210 cells

The effects of the dialdehyde derivatives of inosine (Inox) and 5′-deoxyinosine (5′-dInox) on L1210 cells were compared. The growth of L1210 cells was inhibited to a greater extent by 5′-dInox than by Inox. The increased inhibition of L1210 cell growth by 5′-dInox was also reflected by the increased inhibition of the incorporation of precursors into RNA, DNA and proteins. Even though 5′-dInox was a more potent inhibitor, Inox accumulated in the L1210 cells to levels 4- to 5-fold greater than 5′-dInox. The metabolism of [5- 3H]deoxycytidine and [5- 3H]deoxyuridine by L1210 cells in culture, in the presence of Inox or 5′-dInox, indicated that dCMP deaminase was an intracellular site of action for 5′-dInox. The dCMP deaminase activity in cell-free extracts prepared from 5′-dInox-treated cells was reduced markedly. This decrease in activity was not reversed by increased substrate concentrations nor was the activity subject to allosteric activation by dCTP. Deoxyuridine and deoxycytidine were able to reverse the effects of 5′-dInox on the inhibition of L1210 cell growth.

DNA-fork displacement rates in cultured mammalian cells with mutations in ribonucleotide reductase

DNA-replication fork displacement rates were measured in mouse S49 lymphosarcoma cell lines and in derivatives of those cell lines. One of the derivatives lacks dCMP deaminase activity and two others bear defined mutations in ribonucleotide reductase. We also examined a revertant cell line that was selected from one of the ribonucleotide reductase mutants and has regained normal ribonucleotide reductase activity. Our results show a correlation between decreased fork-displacement rates and alterations in ribonucleotide reductase, suggesting a possible involvement of this enzyme in the replication apparatus.

Mechanism of UV-induced deoxynucleoside triphosphate pool imbalance in CHO-K1 cells

Several laboratories have reported that exposure of cells to UV radiation results in a significant imbalance in deoxynucleoside triphosphate pool concentrations. In our CHO-K1 cells, a rapid drop in dCTP is accompanied by a rapid increase in dTTP. Examination of enzyme activities associated with synthesis/degradation of these molecules suggests that UV transiently enhances a putative dCTPase, dCMP deaminase and CdR kinase activities. This results in accumulation of excess dUMP which is probably converted to dTMP, then to dTTP. The absence of dCMP deaminase in V79 cells prohibits this rapid response in those cells. Moreover, significantly different dCMP deaminase activities were observed in CHO-K1 cells obtained from other laboratories, suggesting they, too, may respond differently to irradiation.

Analysis of the drug synergism between thymidine and arabinosyl cytosine using mouse S49 T lymphoma mutants.

The synergism between arabinosyl cytosine (araC) and thymidine is characterized using two mutant S49 T lymphoma cell populations with altered deoxyribonucleotide metabolism. AraC-1-6 cells are deficient in dCMP deaminase activity resulting in a secondary elevation of intracellular dCTP pools, whereas dGuo-200-1 cells have a mutation in the Ml subunit of ribonucleotide diphosphate reductase, which also results in elevation of dCTP levels. These two mutant cell populations are partially resistant to araC cytotoxicity as compared to the wild type cells. The resistance to araC is contributed to the elevation of dCTP levels in these mutants which prevent araC incorporation into the DNA due to feedback inhibition of deoxycytidine kinase. Addition of extracellular thymidine to dCMP deaminase deficient cells causes a decrease in dCTP levels and in parallel increase their sensitivity to araC. In contrast, extracellular thymidine does not reduce dCTP levels in the mutant cells with altered ribonucleotide reductase and no synergism between araC and thymidine is observed in these cells. The expansion of dTTP pools in the presence of thymidine is similar in the two mutants. These results suggest that the depletion of dCTP pools by thymidine is responsible for the synergistic action of thymidine on araC cytotoxicity and that dTTP does not directly enhance the incorporation of araC into the DNA of T lymphoma cells.

Deoxyribonucleoside triphosphate metabolism and the mammalian cell cycle: Effects of thymidine on wild-type and dCMP deaminase-deficient mouse S49 T-lymphoma cells

The size of the dCTP pool has been implicated as a possible regulator of DNA synthesis. In this investigation we correlate large intracellular variations in deoxyribonucleoside triphosphate levels to the growth rates and cell-cycle kinetics of mouse S49 T-lymphoma cells. Wild-type and a mutant line AzidoC-100-5, lacking dCMP-deaminase activity resulting in a 10-fold expanded dCTP pool were studied and compared using flow cytometry, centrifugal elutriation and nucleoside triphosphate determinations. An increase in the dCTP pool was closely correlated to the passage of cells from G1 to S phase in both cell types. Addition of thymidine to wild-type and mutant cells resulted in an accumulation of cells in early S phase, concomitant with a decreased dCTP level. Mutant cells excreted large amounts of deoxycytidine into the medium which partially protected the cells from thymidine inhibition. The doubling times for the mutant and wild-type cells were very similar but the mutant had a somewhat prolonged S phase and shortened G1 phase compared with the wild-type cells. Large changes in the DNA precursor levels were produced by addition of thymidine to mutant cultures. This gave no change in the growth rate but a somewhat shortened S phase and prolonged G1. The biochemical background for these effects is discussed.

Pleiotropic mutants of Chinese hamster cells with altered cytidine 5'-triphosphate synthetase.

Following chemical mutagenesis and multiple-step indirect selection, four clones of Chinese hamster V79 cells were isolated which exhibited auxotrophy for thymidine, deoxycytidine, or deoxyuridine but not for cytidine or uridine. All were resistant to uridine, 3-deazauridine, 5-fluorouridine, thymidine, and cytosine arabinoside at concentrations that were toxic to wild-type V79 cells. The cytidine 5'-triphosphate (CTP) and deoxycytidine 5'-triphosphate (dCTP) pools in the mutants were expanded, but the uridine 5'-triphosphate (UTP) pool either decreased or remained unchanged relative to the wild-type level. Furthermore, since the parental cells appear to be deficient in dCMP deaminase activity and CTP (or one of its metabolites) has been shown to inhibit uridine 5'-diphosphate (UDP) reduction, an elevated CTP level should lead to the observed thymidine auxotrophy. It also explains the joint resistance of mutant clones to thymidine and cytosine arabinoside. The change in the ratio of intracellular dCTP to thymidine 5'-triphosphate (dTTP) may be responsible for the elevation in the rates of spontaneous mutations in these mutants.

Isolation of a Saccharomyces cerevisiae mutant strain deficient in deoxycytidylate deaminase activity and partial characterization of the enzyme.

Deoxycytidylate deaminase activity in Saccharomyces cerevisiae has been partially characterized. The yeast enzyme was found to exhibit properties similar to those of dCMP deaminases isolated from higher eucaryotes. A mutant strain completely deficient in dCMP deaminase activity was isolated by selection for resistance to 5-fluoro-2'-deoxycytidylate followed by screening for cross sensitivity to 5-fluoro-2'-deoxyuridylate, a potent inhibitor of the yeast thymidylate synthetase. We have designated this new allele dcd1 . A strain exhibiting an auxotrophic requirement for dUMP was isolated after mutagenesis of a dcd1 tup7 haploid. Genetic analysis revealed that this auxotrophic phenotype resulted from a combination of the dcd1 allele and a second, unlinked, nuclear mutation that we designated dmp1 . This allele, which by itself conveys no readily discernible phenotype, presumably impairs efficient synthesis of dUMP from UDP. The auxotrophic requirement of dcd1 dmp1 tup7 strains also can be satisfied by exogenous dTMP but not deoxyuridine.