Deoxycytidine Triphosphatase Research Articles

The First Structure of an Active Mammalian dCTPase and its Complexes With Substrate Analogs and Products

Precise regulation of dNTPs within the cellular nucleotide pool is essential for high accuracy of DNA replication and is critical for retaining the genomic integrity. Recently, human dCTPase (deoxycytidine triphosphatase), also known as DCTPP1 (human all-alpha dCTP pyrophosphatase 1), has been revealed to be a key player in the balance of pyrimidine nucleotide concentrations within cells, with DCTPP1 deficiency causing DNA damage and genetic instability in both chromosomal and mitochondrial DNA. DCTPP1 also exhibits an additional “house cleaning” function as it has been shown to be highly active against modified cytidine triphosphates, such as 5-methyl-dCTP, which, if incorrectly incorporated into DNA can introduce undesirable epigenetic marking. To date, structural studies of mammalian dCTPase have been limited to inactive constructs, which do not provide information regarding the catalytic mechanism of this important enzyme. We present here the first structures of an active mammalian dCTPase from M. musculus in complex with the nonhydrolyzable substrate analog dCMPNPP and the products 5-Me-dCMP and dCMP. These structures provide clear insights into substrate binding and catalysis and clearly elucidate why previous structures of mammalian dCTPase were catalytically inactive. The overall structure of M. musculus dCTPase is highly similar to enzymes from the all-alpha NTP phosphohydrolase superfamily. Comparison of M. musculus dCTPase with homologs from a diverse range of mammals, including humans, shows that the residues, which contribute to substrate recognition, are entirely conserved, further supporting the importance of this enzyme in the protection of genomic integrity in mammalian cells.

Pyrimidine Deoxyribonucleotide Metabolism in Members of the Class Mollicutes

Cell extracts from six Acholeplasma species, six Mycoplasma species, and Spiroplasma floricola 23-6T (T = type strain) were examined for enzyme activities of pyrimidine deoxyribonucleotide metabolism. All of these organisms had thymidine kinase and thymidine phosphorylase activities, and all lacked deoxycytidine triphosphatase activity. The 13 members of the Mollicutes were separated into three groups by the presence or absence of the following four enzyme activities: (i) the adenosine triphosphate-insensitive deoxyuridine triphosphate-specific hydrolyzing deoxyuridine triphosphatase, (ii) a deoxyuridine monophosphate phosphatase, (iii) deoxycytidine deaminase, and (iv) deoxycytidine monophosphate deaminase. Five of the six Acholeplasma species (all Acholeplasma species except Acholeplasma florum L1T) had all four enzymatic activities. The six Mycoplasma species only had the deoxycytidine and deoxycytidine monophosphate deaminase activities. The only two plant isolates studied, A. florum L1T and S. floricola 23-6T, lacked all four enzymatic activities.

Synthesis of functional bacteriophage T4-delayed early mRNA in the absence of protein synthesis

When Escherichia coli B207 is grown either aerobically or under limited aerobic conditions, pretreated with chloramphenicol to block protein synthesis, and then infected with bacteriophage T4, the phage RNA which accumulates, termed "immediately early" (IE), contains the transcripts of a limited number of prereplicative genes. Among the transcripts which accumulate is the mRNA which serves as a template for deoxycytidylate hydroxymethylase (HMase) synthesis. Among the prereplicative gene transcripts which do not accumulate under these conditions are deoxycytidine triphosphatase (dCTPase), alpha-glucosyl transferase (alphg-gt), and deoxynucleotide kinase (kinase); these genes have been termed "delayed early" (DE). In contrast, when protein synthesis is inhibited by depleting aerobically grown E. coli B207 of K+, both IE and DE T4 RNA accumulate, but these transcripts do not contain functional HMase, dCTPase, alpha-gt, or kinase mRNA's. However, if E. coli is grown under conditions of limited aeration and then depleted of K+ prior to T4 infection, the T4 RNA which accumulates contains both IE and DE transcripts and functional HMase, dCTPase, and alpha-gt mRNA's. Functional kinase mRNA does not accumulate under these conditions. The results of these experiments indicate that the synthesis of functional DE RNA in the absence of simultaneous protein synthesis, depends on the physiological condition of the cells and the way in which protein synthesis is inhibited. In addition, data is presented which suggests that extensive transcription of DE genes in the absence of protein synthesis results in the inhibition of transcription of certain IE genes.

Effect of chloramphenicol and starvation for an essential amino acid on the synthesis and decay of T4 bacteriophage-specific messengers transcribed from early and quasi-late promoters

Results of experiments in which the interval between infection and addition of rifampicin was varied indicated that messengers for T4 phage-specific dihydrofolate reductase, dCMP hydroxymethylase, both glucosyltransferases, DNA methylase, dCMP deaminase, and deoxycytidine triphosphatase are transcribed uniquely from early promoters (i.e., promoters that start to function moments after infection). In contrast, messengers for deoxynucleoside monophosphate kinase are transcribed exclusively from a quasi-late promoter (i.e., a promoter that does not function until 2 min postinfection). Messengers for DNA polymerase and dTMP synthetase are transcribed from both kinds of promoters. Transcription from early (P E) and quasi-late (P Q) promoters was studied under conditions that limit phage-specific protein synthesis to 5 or 10% of its normal maximum value. The conditions employed were treatment with chloramphenicol and starvation for an essential amino acid (in this case, leucine). Accumulation of viral-coded messengers was monitored by the ability of RNA made in vivo to sustain specific enzyme synthesis when rifampicin, a potent inhibitor of the initiation of RNA synthesis, was added prior to the restoration of protein synthesis. During treatment with chloramphenicol or starvation for an essential amino acid, RNA chains were initiated exclusively at P E'S. However, chloramphenicol limited transcription to presumed P E-proximal genes whereas P E-proximal and distal genes were transcribed during starvation for an essential amino acid. Under both conditions, the transcriptive yield from P E's was subnormal. In leucine-supplemented hosts, P E-distal and P Q transcription became chloramphenicol-refractile by the second minute. On the other hand, P E-distal transcription remained chloramphenicol-sensitive until the fourth minute in leucine-depleted hosts. Global T4 prereplicative messengers decayed with a functional half-life of 8–9 min in both leucine-supplemented and starved hosts. In both cases, decay followed simple exponential kinetics until, at least, 1% survival. Chloramphenicol stabilized most messengers in both supplemented and starved hosts. When chloramphenicol was added 4 min after infection of leucine-supplemented hosts, messengers transcribed from P E's were stablized but messengers transcribed from P Q's were not. About 50% of the decay of P Q-initiated messengers in chloramphenicol was due to the suA nuclease. The data are discussed in terms of the interrelationship between postinfection protein synthesis and prereplicative transcriptive events in bacteriophage T4.

Characterization of new regulatory mutants of bacteriophage T4.

Plating techniques which eliminate T4 plaque formation on Escherichia coli by folate analogue inhibition of dihydrofolate (FH(2)) reductase (EC 1.5.1.3) allowed the isolation of folate analogue-resistant (far) mutants of T4. One class of far mutants overproduces the phage-induced FH(2) reductase. Deoxycytidylate deaminase (EC 3.5.4.12), thymidine kinase (EC 2.7.1.21), and deoxycytidine triphosphatase (EC 3.6.1.12) are also overproduced by 20 min after infection at 37 C. The overproduction of FH(2) reductase by these far mutants is not affected by the absence of DNA synthesis. Other types of mutations that affect the synthesis of early enzymes cause overproduction in the absence of DNA synthesis of some of the above enzymes but not of FH(2) reductase. Therefore, overproducing far mutants apparently have mutations in previously undescribed genes controlling the expression of the T4 genome. Three of four mutants under study map near gene 56, and one maps near gene 52. All of these mutants show delays in DNA synthesis, phage production, and lysis and appear to show decreased levels of RNA synthesis based on the cumulative incorporation of uridine.

Phenotypic reversion by 5-fluorouracil of Amber mutations in bacteriophage T4: Characterization of rescued functions

Phenotypic reversion by 5-fluorouracil of amber mutations in phage T4 was studied. The rescued functions of the early genes 1 (deoxynucleotide kinase), 42 (deoxycytidylate hydroxymethylase) and 56 (deoxycytidine triphosphatase) were investigated by direct measurements of the rescued enzyme activities. Addition of 5-fluorouracil early (2 min after infection) but not late (16 min) was effective in rescuing enzyme activities corresponding to the mutated genes. The time course of formation of the rescued enzymes was similar to that of other early enzymes in nonpermissive bacteria after DNA-negative mutant infection: Enzyme activity increased for 10–15 min beyond the time of shut-off in wild-type T4-infected cells. These results indicate that transcription of the mutated early genes, rather than the translation of early messengers, is restricted in the late period of infection with early amber mutants.

Control of T4 endonuclease IV by the D2a region of bacteriophage T4

An rII deletion mutant of bacteriophage T4 in which the deletion extends into the adjacent D2a region fails to induce detectable amounts of T4 endonuclease IV. Since the same mutant was shown by others to lead to anomalous synthesis of DNA when coupled to a mutation in gene 56 (deoxycytidine triphosphatase), T4 endonuclease IV may have a role in degrading newly replicated cytosine containing DNA. It is uncertain whether the D2a region contains the structural gene for T4 endonuclease IV.

Direct participation of dCMP hydroxymethylase in synthesis of bacteriophage T4 DNA.

In order to retain in an in situ system the control mechanisms involved in synthesis of bacteriophage T4 DNA, infected cells were made permeable to nucleotides by plasmolysis with concentrated sucrose. Such preparations use exogenous deoxyribonucleotides to synthesize T4 phage DNA. As has been observed with in vivo studies, DNA synthesis was drastically reduced in plasmolyzed preparations from cells infected by amber mutants of genes 1, 32, 41, 42, 43, 44, or 45. Added 5-hydroxymethyl dCTP did not bypass either a mutant of gene 42 (dCMP hydroxymethylase) or of gene 1 (phage-induced deoxyribonucleotide kinase). In a phage system lacking deoxycytidine triphosphatase (gene 56) and the gene-46 product, and therefore incorporating dCTP into DNA, dCTP incorporation did not require dCMP hydroxymethylase, in keeping with in vivo results. With a triple amber mutant of genes 1, 46, and 56 only slight incorporation of dCTP occurred. By contrast, in experiments performed in vivo the synthesis of cytosine-containing DNA was unaffected by an amber mutation in gene 1. These studies provide evidence that dCMP hydroxymethylase, in addition to its known catalytic function, has a second, more direct, role in phage T4 DNA synthesis, apparently in recognition of hydroxymethyl dCTP. The role of the phage-induced deoxyribonucleotide kinase in T4 DNA synthesis in the plasmolyzed system remains unresolved.

Degradation of Escherichia coli chromosome after infection by bacteriophage T4: role of bacteriophage gene D2a.

Mutations in the D2a gene of bacteriophage T4 have recently been shown to result in the stabilization of cytosine-containing phage deoxyribonucleic acid (DNA) made after infection by phage gene 56 (deoxycytidine triphosphatase) mutants. In the experiments reported here, we investigate the role of the D2a gene in the degradation of the host chromosome. We find that if T4 endonuclease II, a product of the phage gene denA, is active, host chromosome degradation appears normal, regardless of the presence of the D2a gene product. However, if T4 endonuclease II is absent, a small amount of host chromosome degradation occurs, but only if the D2a product is present. These results are interpreted in terms of the hypothesis that D2a controls a nuclease which degrades cytosine-containing DNA. Neither D2a nor denA mutations affect the shut-off of host DNA synthesis.

Methylation of adenine residues in bacteriophage T2 DNA containing cytosine in place of 5-hydroxymethylcytosine

It has been previously shown that uP1 mutants, derived from phage T2 gt rP1, determine an altered form of the phage-induced DNA methylase. The uP1 enzyme methylates two- to threefold more adenine residues on phage DNA than does the wild-type methylase. The question of whether the presence of 5-hydroxymethylcytosine (HMC) in T2 DNA plays a role in determining methylase specificity was investigated. A triple amber mutant was constructed which is defective in the synthesis of the enzymes, deoxycytidine triphosphatase (dCTPase) and deoxycytidylate hydroxymethylase (dCMP-HMase) as well as in the ability to degrade completely host DNA (genes 56, 42, and 47, respectively). In host cells lacking an amber suppressor ( su −), the mutant is shown to synthesize DNA which contains cytosine (C) in place of HMC. This C-containing DNA has the same level of N 6-methyladenine (MeAde) as observed in the HMC-containing DNA of T2 gt rP1; in contrast, the HMC-containing DNA produced by T2 gt uP1 phage had two- to threefold more MeAde. Furthermore, the T2 am 42, 47, 56 and T2 gt rP1 DNAs behaved similarly as substrates for in vitro methylation by extracts of phage-infected cells. These data indicate that the 5-hydroxymethyl group on HMC does not interfere with the activity of the wild-type DNA methylase, nor does it enhance the activity of the uP1 enzyme.

Stability of cytosine-containing deoxyribonucleic acid after infection by certain T4 rII-D deletion mutants.

When T-even phage infect Escherichia coli, synthesis of host deoxyribonucleic acid (DNA) rapidly ceases. If the phage carry a mutation in a gene essential for phage DNA synthesis, then the infected bacteria should make no DNA, either host DNA or phage DNA. However, we have found that infection with certain T4 gene 56 (deoxycytidine triphosphatase)-rII double mutants leads to substantial DNA synthesis. Only rII deletion mutations which extend into the middle third of the adjacent, nonessential D region lead to the anomalous DNA synthesis, when combined with a gene 56 mutation; the requirement probably is that the deletion extend into the D2a transcriptional unit identified by Sederoff et al. Genetic evidence indicates that the observed anomalous DNA synthesis is synthesis of phage DNA. We suggest that the D2a region controls, directly or indirectly, a nuclease involved in the breakdown of cytosine-containing DNA. In the absence of the D2a product, the cytosine-containing phage DNA made by the gene 56 mutant is stabilized.

Properties of phage T4 messenger RNA synthesized in the absence of protein synthesis

Phage T4 specific messenger RNA (mRNA) synthesized by Escherichia coli B cells infected in the absence of protein synthesis was studied. Protein synthesis was inhibited by treatment of bacterial cells with the antibiotics chloramphenicol (CM), puromycin (PM), or amicetin (AMI) or by K + depletion of the K + accumulation defective mutant strain B207. As shown by hybridization competition experiments only “immediate early” mRNA was made in the presence of CM or in the absence of K +. When inhibition of protein synthesis was released and further RNA synthesis was blocked with rifampicin (RIF), the immediate early mRNA accumulated either in the presence of CM, PM, or AMI or in the absence of K + could serve as messenger for the synthesis of the protein necessary for resistance to lysis from without, but could not serve as messenger for the synthesis of two early phage enzymes, deoxynucleotide kinase (TMP kinase) and deoxycytidine triphosphatase (dCTPase). RNA synthesized in the presence of CM could serve as mRNA for the synthesis of the early enzyme deoxycytidylate hydroxymethylase (HMase). However, RNA synthesized in the absence of K + or in the presence of PM or AMI could not direct the synthesis of this enzyme. The results indicate that although phage mRNA is made in the absence of concomitant protein synthesis, it contains only a very restricted portion of the RNA made during a normal infection. Furthermore, the RNA made in the presence of CM is not identical with that synthesized during inhibition of protein synthesis by the other methods employed.

Formation of RNA in T4 phage-infected bacteria: Transcriptional regulation of early enzyme synthesis

The rates of synthesis were determined for those early mRNA species which code for early enzymes involved in phage-specific DNA synthesis. Evidence was found for a regulation mechanism which specifically restricts the transcription of early genes. The formation of early mRNA representing certain early genes was measured at different times after infection by phenotypic suppression by 5-fluorouracil of amber mutations in the early genes 1 (hydroxymethyl deoxycytidylate kinase), 42 (deoxycytidylate hydroxymethylase), and 56 (deoxycytidine triphosphatase), respectively. Suppression was measured by the determination of DNA synthesis and phage production. Since 5-fluorouracil suppression of amber mutations is effected by the incorporation of the analog into mRNA, the relative efficiency of suppression at different times after infection was interpreted to reflect the rate of mRNA formation at the time of fluorouracil addition. The regulation of early mRNA synthesis was also directly demonstrated by short-time labeling with uracil- 3H and fluorouracil- 3H after the infection of E. coli B with the mentioned phage mutants, which are DNA negative and produce no late mRNA. The uptake of 5-fluorouracil as an RNA precursor into the ribonucleotide pools of the infected cell was checked by electrophoretic analysis at different times after infection.

Regulation of cessation of early enzyme synthesis in bacteriophage T4 infected Escherichia coli: Separate mechanisms for different enzymes

The kinetics of synthesis of four early enzymes (deoxycytidine triphosphatase, deoxycytidylate hydroxymethylase, deoxynucleotide kinase and thymidylate synthetase) were studied in bacteriophage T4-infected Escherichia coli B207. E. coli B207 is a mutant of E. coli B which is defective in the accumulation and retention of K+ from the growth medium. In mutant cells depleted of K+, the rate of protein synthesis can be regulated by the level of this cation in the growth medium. When bacteriophage T4 infected E. coli B207 cells were incubated in normal growth medium (i.e., containing 33 m M K+), the kinetics of synthesis of the four enzymes studied were identical. However, when infected B207 cells were allowed to synthesize protein at 50% the normal rate (in medium containing 1 m M K+) it was possible to subdivide the four early enzymes into three separate classes based on their kinetics of synthesis. Deoxycytidine triphosphatase and deoxycytidylate hydroxymethylase proved to be members of the same class of early enzyme, while deoxynucleotide kinase and thymidylate synthetase were found to be members of a second and third class, respectively.

Biological effects of substituting cytosine for 5-hydroxymethylcytosine in the deoxyribonucleic acid of bacteriophage T4.

Previous work from this laboratory has shown that the cytosine-containing T4 deoxyribonucleic acid (DNA) made by deoxycytidine triphosphatase (dCTPase) amber mutants is extensively degraded, and that nucleases controlled by genes 46 and 47 participate in this process. In this paper, we examine other consequences of a defective dCTPase. Included are studies of DNA synthesis and phage production, and of the control of both early and late protein synthesis after infection of Escherichia coli B with various T4 mutants defective in genes 56 (dCTPase), 42 (dCMP hydroxymethylase), 1 (deoxynucleotide kinase), 43 (DNA polymerase), 30 (polynucleotide ligase), 46 and 47 (DNA breakdown) or e(lysozyme). By varying the temperature of infection with a temperature-sensitive dCTPase mutant, we have been able to control intracellular dCTPase activity, and thus vary the cytosine content of the phage DNA. We have produced and characterized viable T4 phage in which cytosine replaces 20% of the 5-hydroxymethylcytosine (HMC) in the DNA. We present evidence which suggests that intact, cytosine-containing T4 DNA is much less efficient than is normal T4 DNA in directing the synthesis of tail-fiber antigen. Lysozyme production is much less affected by progressively decreasing dCTPase activity; however, complete substitution of cytosine is correlated with a depression of lysozyme synthesis greater than expected from the defective synthesis of DNA. Low but significant lysozyme synthesis is observed late after infection of E. coli B with T4 amber mutants defective in a number of genes controlling DNA synthesis. The "20% cytosine" T4 phage, once produced, can initiate an apparently normal infection at permissive temperatures; the synthesis of early enzymes, DNA, and phage does not appear to be impaired. Two roles for HMC in T4 DNA have been indicated previously: (i) involvement in host-controlled restriction of the phage, in which glucosylation of the hydroxymethyl group plays a crucial role (16, 29, 53, 58), and (ii) protection of vegetative DNA against phage-controlled nucleases, a protection not dependent on glucosylation (41, 66, 67). A third role is suggested by our present results: transcription of at least some late genes can occur only from HMC-containing DNA and not from cytosine-containing DNA.

Evidence that gene 56 of bacteriophage T4 is a structural gene for deoxycytidine triphosphatase

Gene 56 of bacteriophage T4 was previously shown to control deoxycytidine triphosphatase (dCTPase), an enzyme known also to dephosphorylate dCDP, dUTP, and dUDP. Whether this control is direct or indirect was not clear. In the present study we examined the heat lability of the dCTPases produced by gene 56 amber mutants in four different amber-suppressor strains. Others have shown that tyrosine and glutamine are inserted at the amber codon by two of these suppressor strains; the other two insert serine. With one mutant, amE51×5, no difference in heat lability from the wild-type enzyme was observed in the enzymes produced by any of the four suppressor strains. With another mutant, amC153×1, a marked increase in heat lability was observed when the suppressor strains were those inserting tyrosine or serine, but not glutamine. These and other observations constitute strong evidence that gene 56 is a structural gene for the enzyme dCTPase.

Protein synthesis by Escherichia coli infected with bacteriophage T4D

Proteins made by E. coli cells infected with bacteriophage T4 were analyzed by a method which combined disc electrophoresis and autoradiography. The precursorproduct relationship between subunits and larger components (head, tail) was studied by taking advantage of the fact that the larger components cannot penetrate into the gel used in disc electrophoresis. These studies have shown that the early proteins are not controlled as a single homogeneous class all members of which are synthesized during the same time periods; instead, they start being formed and are shut off at various times during the early part of the infection process. Amber mutants in gene 30 (polynucleotide ligase-defective) synthesized a small amount of DNA, which was later degraded to a fraction soluble in trichloroacetic acid. The ligase-defective mutants were capable of synthesizing an almost normal amount of late proteins, whereas all other DNA-negative mutants, including a deoxycytidine triphosphatase(dCTPase)-defective mutant and maturation-defective mutants could not induce late protein synthesis. The dCTPase-defective mutant, which also synthesizes a small amount of unstable DNA, did not induce late protein synthesis even when the degradation of DNA was prevented.

Repair and recombination in phage T4. II. Genes affecting UV sensitivity.

The effect of temperature sensitive (ts) mutations in five early function genes on the sensitivity of phage T4D to ultraviolet (UV) light was determined. Temperature sensitive mutations in genes 30, 43, and 47 were found to increase the sensitivity of T4D to ultraviolet light. Mutations in genes 45 and 56 did not alter UV sensitivity. Gene 30 has been identified as the structural gene for polynucleotide ligase (Fareed and Richardson, 1967); gene 43 codes for DNA polymerase (deWaard et al., 1965; Warner and Barnes, 1966); mutants in gene 47 are unable to degrade the host chromosome (Wiberg, 1966) and are probably defective in a deoxyribonuclease. Deoxycytidine triphosphatase is the enzyme specified by gene 56 (Wiberg, 1967); and the product of gene 45 has not been identified.

Amber Mutants of Bacteriophage T4 Defective in Deoxycytidine Diphosphatase and Deoxycytidine Triphosphatase: ON THE ROLE OF 5-HYDROXYMETHYLCYTOSINE IN BACTERIOPHAGE DEOXYRIBONUCLEIC ACID

Infection of Escherichia coli B with T4 amber mutants in gene 56 fails to cause the appearance of either deoxycytidine triphosphatase or deoxycytidine diphosphatase, and little or no DNA synthesis results. Infection of E. coli B with a 7:3 mixture of a gene 56 amber mutant and wild type T4 results in the appearance of only 30% as much dCTPase activity as normal but gives a 100% yield of mixed phage. This indicates that dCTPase is present in at least a 3-fold excess in the wild type infection. Escherichia coli W4597, shown by others to support the synthesis by wild type T4 of DNA in which the hydroxymethylcytosine is virtually unglucosylated, also fails to support the synthesis of DNA upon infection with a gene 56 amber mutant. This and other evidence indicate that in T4 DNA a crucial function of hydroxymethylcytosine, whether glucosylated or not, is the protection of progeny phage DNA against nuclease activity that degrades the cytosine-containing DNA of the host.

Bacteriophage-induced functions in Escherichia coli K (lambda) infected with rII mutants of bacteriophage T4.

Rutberg, Blanka (Karolinska Institutet, Stockholm, Sweden), and Lars Rutberg. Bacteriophage-induced functions in Escherichia coli K(lambda) infected with rII mutants of bacteriophage T4. J. Bacteriol. 91:76-80. 1966.-When Escherichia coli K(lambda) was infected with rII mutants of phage T4, deoxycytidine triphosphatase, one of the phage-induced early enzymes, was produced at initially the same rate as in r(+)-infected cells. Deoxyribonuclease activity was one-third to one-half of that of r(+)-infected cells. This lower deoxyribonuclease activity was observed also in other hosts or when infection was made with rI or rIII mutants. Presence of chloramphenicol did not allow a continued synthesis of phage deoxyribonucleic acid in rII-infected K(lambda). No phage lysozyme was detected nor was any antiphage serum-blocking antigen found in rII-infected K(lambda). It is suggested that the rII gene is of significance for the expression of phage-induced late functions in the host K(lambda).