DNA nucleotide excision-repair synthesis is dependent of pertubations of deoxynucleoside triphosphate pool size

We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [³H]TTP from [³H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [³H]TTP pool was formed within 30 min of the addition of [³H]thymidine and remained relatively constant for the next 6 h. Addition of 2–10 mM hydroxyurea (HU) caused a gradual 2–4-fold increase in the [³H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [³H]TTP pool size in cells exposed to 20 M/m² and unrradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8–10-fold reduction in the size of the [³H]TTP pool relative to the initial studies. In the UV excision-repair studies the precense of hydroxyurea did not alter the specific activity of [³H] thymidine 5'-monophospahte incorporated into parental DNA due to repaier replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair sythesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [³H]TTP pool.     

Inhibitors of ribonucleotide reductase alter DNA repair in human fibroblasts through specific depletion of purine deoxynucleotide triphosphates

Hydroxyurea, deoxyadenosine, pyridine-2-carboxaldehyde thiosemicarbazone, pyrazoloimidazole, 3,5-diamino-1,2,4 triazole (guanazole), 3,4,5-trihydroxy benzohydroxamic acid and 3,4-dihydroxy benzohydroxamic acid were examined for their effects on cellular dNTP pools, DNA excision repair, DNA replication and deoxynucleoside uptake in human diploid fibroblasts. All 7 agents were effective inhibitors of the UV excision repair process in noncycling quiescent cells, but not in rapidly dividing log-phase cells. This differential effect clearly demonstrates dependency upon modulation of cellular purine dNTP pool levels at the level of the reductase. Repair synthesis is shown to be less sensitive to all 7 reductase inhibitors than is replicative synthesis. Studies on cellular uptake of labeled DNA precursors in inhibitor-treated cells support the notion that deoxynucleosides cannot channel into the replicative synthesis process whereas they are readily utilized at repairing sites.Abbreviations HU hydroxyurea - dA deoxyadenosine - TSC pyridine-2-carboxaldehyde thiosemicarbazone - IMPY pyrazoloimidazole - THBA 3,4,5-trihydroxy benzohydroxamic acid - DHBA 3,4-dihydroxy benzohydroxamic acid - UDS unscheduled DNA synthesis - dT thymidine - dNTP deoxynucleoside triphosphate     

Relations between synthesis of deoxyribonucleotides and DNA replication in 3T6 fibroblasts

In exponentially growing 3T6 cells, the synthesis of deoxythymidine triphosphate (dTTP) is balanced by its utilization for DNA replication, with a turnover of the dTTP pool of around 5 min. We now investigate the effects of two inhibitors of DNA synthesis (aphidicolin and hydroxyurea) on the synthesis and degradation of pyrimidine deoxynucleoside triphosphates (dNTPs). Complete inhibition of DNA replication with aphidicolin did not decrease the turnover of pyrimidine dNTP pools labeled from the corresponding [3H]deoxynucleosides, only partially inhibited the in situ activity of thymidylate synthetase and resulted in excretion into the medium of thymidine derived from breakdown of dTTP synthesized de novo. These data demonstrate continued synthesis of dTTP in the absence of DNA replication. In contrast, hydroxyurea decreased the turnover of pyrimidine dNTP pools 5-50-fold. Hydroxyurea is an inhibitor of ribonucleotide reductase and stops DNA synthesis by depleting cells of purine dNTPs but not pyrimidine dNTPs. Our results suggest that degradation of dNTPs is turned off by an unknown mechanism when de novo synthesis is blocked.     

The role of deoxynucleoside triphosphate pools in the inhibition of DNA-excision repair and replication in human cells by hydroxyurea

The effects of hydroxyurea (HU) on the DNA-excision repair process in human cells has been systematically examined. It is demonstrated that HU induces DNA single-strand break accumulation in a dose-dependent fashion in ultraviolet-irradiated and MMS-treated confluent but not log-phase fibroblasts and that these breaks are clearly the consequence of the inhibition by HU of the enzyme, ribonucleotide reductase. The breaks form rapidly, are stable for at least 10 h and largely disappear by 20 h. The production of these DNA-strand breaks is antagonized by a combined treatment of 10 microM deoxyadenosine, deoxycytidine and deoxyguanosine whereas thymidine potentiates strand-break formation at low HU concentrations. It is also confirmed that HU, while inhibiting replicative synthesis has no apparent inhibitory effect on unscheduled DNA synthesis (UDS) although the increased uptake of labeled DNA precursors into HU-treated cells makes it difficult to assess the actual effects on the repair-synthetic process. Analysis of the effects of HU on deoxynucleoside triphosphate pool levels and the demonstration of the failure of the HU block to replicative synthesis to be reversed by high (1 mM) concentrations of added deoxynucleosides lend support to the notion of compartmentalized dNTP pools for repair and replication.     

Changes of deoxyribonucleoside triphosphate pools induced by hydroxyurea and their relation to DNA synthesis

Hydroxyurea inactivates ribonucleotide reductase from mammalian cells and thereby depletes them of the deoxynucleoside triphosphates required for DNA replication. In cultures of exponentially growing 3T6 cells, with 60-70% of the cells in S-phase, 3 mM hydroxyurea rapidly stopped ribonucleotide reduction and DNA synthesis (incorporation of labeled thymidine). The pool of deoxyadenosine triphosphate (dATP) decreased in size primarily, but also the pools of the triphosphates of deoxyguanosine and deoxycytidine (dCTP) were depleted. Paradoxically, the pool of thymidine triphosphate increased. After addition of hydroxyurea this pool was fed by a net influx and phosphorylation of deoxyuridine from the medium and by deamination of intracellular dCTP. An influx of deoxycytidine from the medium contributed to the maintenance of intracellular dCTP. 10 min after addition of hydroxyurea, DNA synthesis appeared to be completely blocked even though the dATP pool was only moderately decreased. As possible explanations for this discrepancy, we discuss compartmentation of pools and/or vulnerability of newly formed DNA strands to nuclease action and pyrophosphorolysis.     

Enhancement of repair replication in mammalian cells by hydroxyurea

The effect of hydroxyurea on DNA repair replication has been studied in Chinese hamster ovary cells. Mitotic cells were treated with UV irradiation, methyl methanesulfonate or nitrogen mustard and incuated in the presence of each of the 4 [³H]deoxyribonucleosides plus BrdUrd and FdUrd for 2 h. The amount of repair replication was quantitated on CsCl gradients and similar values were obtained for each nucleoside. In all cases addition of HU during the incubation period increased these values approximately 2-fold. Following MMS treatment, pool sizes for each of the nucleosides were estimated by varying the amount of exogenously supplied nucleoside. They were found to be insensitive to the addition of HU and it is concluded that the increased incorporation of [³H]deoxyribonucleosides in the presence of HU reflects an increased amount of repair replication.     

Simultaneous detection of DNA strand breaks and unscheduled DNA synthesis in mutagen-treated human lymphocytes in the absence of hydroxyurea

Human lymphocytes in the quiescent state were exposed to UVC radiation. After irradiation the cells were allowed to repair for various times in the presence of [3H]thymidine or [3H]deoxycytidine in the culture medium. Hydroxyurea was not used to suppress semiconservative DNA replication in the small number of growing cells. After incubation DNA strand breaks were detected by the DNA-unwinding method and the amount of 3H incorporation in DNA was measured by liquid scintillation counting. The results show that the yield of DNA strand breaks and the amount of unscheduled DNA synthesis (UDS) can be measured from the same lymphocyte sample. A low background 3H incorporation in untreated cells could be achieved even in the absence of hydroxyurea. This requires, however, that 3H incorporation is measured only in the double-stranded DNA and that [3H]dCyd is used instead of [3H]dThd as the labelled deoxynucleoside.     

Effects of DNA replication inhibitors on UV excision repair in synchronised human cells.

When HeLa cells are irradiated with UV and treated with the DNA synthesis inhibitors hydroxyurea (HU) and 1-beta-D-arabinofuranosylcytosine (ara C), DNA strand breaks accumulate at sites where excision repair of DNA damage has been inhibited after the incision step. This break accumulation occurs in mitotic, G1 and S phase cells. But UV-induced repair synthesis of DNA, as measured by [3H]thymidine incorporation into unreplicated DNA, is not inhibited by HU and ara C in G1 or S phase cells, even though replicative synthesis is virtually abolished. Repair and replication must therefore utilise different DNA precursor pools, or different DNA synthetic systems; and the action of Hu and ara C in causing strand break accumulation may occur at the ligation step of excision repair.     

The scintillometric evaluation of DNA repair synthesis can be distorted by changes of thymidine pool radioactivity

The induction of DNA repair synthesis by UV radiation and methylmethane sulphonate (MMS) in mammalian cell lines of human (EUE, HeLa, FT, KB) and hamster (CHO, BHK) origin has been evaluated by means of autoradiography and the scintillometric procedure which implied the use of hydroxyurea (HU) to suppress DNA replication.While with UV radiation both methods produce concordant positive results, in the case of MMS the evidence of DNA repair synthesis obtained from the autoradiograms is occasionally accompanied by a lack of increase of DNA radioactivity in the treated cultures, as detected by scintillation counting. In such instances MMS is shown to reverse the enhancement of pool radioactivity in the cultures incubated with HU and even to reduce the radioactivity of thymidine pool below control values. By normalizing DNA radioactivities on the basis of pool variations, the discrepancy between autoradiography and scintillation counting is solved.The chromatographic analysis of thymidine pool components justifies the normalization procedure as it demonstrates that also in cultures treated with MMS or MMS + HU pool variations closely parallel the variations of thymidine triphosphate (dTTP) level.The normalization of DNA radioactivities based on the overall pool radioactivities gives an improved evaluation of the actual rate of DNA synthesis. It can be recommended for screening studies of DNA repair inducers because it allows one to correct false negative results without producing false positive data. Compared with the dTTP levels, overall pool radioactivities used as normalizing factors still produce an underestimate of DNA repair when high doses of MMS are applied to hamster cell cultures.     

Thymidine triphosphate synthesis in senescent WI38 cells : Relationship to loss of replicative capacity

The effect of in vitro age on thymidine triphosphate (TTP) synthesis was assessed in WI38 cultures according to the following measurements: (1) thymidine kinase activity of broken cell preparations; (2) in situ incorporation of [3H]thymidine into acid-soluble material; and (3) total intracellular TTP content as determined by an enzymatic assay. All three parameters were maximal in exponentially proliferating populations and minimal in quiescent monolayers; no significant differences between young and old cultures were observed despite the reduced replicative capacity of the latter. The addition of serum to density-arrested cultures induced both TTP synthesis and DNA replication after a lag of approx. 12 h; although a greater percentage of young cells initiated replication as compared with old, pool sizes expanded to a similar extent in both populations. Pool expansion did not require entry into S phase; the pool sizes of control and cytosyl arabinoside-treated cultures were comparable. These findings suggest that senescent cells retain the ability to synthesize TTP, even though they are incapable of replicating DNA. Because TTP synthesis is a cell cycle-dependent event that normally begins in late G1, senescent cells might be blocked in the latter portion of the prereplicative phase and not in G0 as are quiescent cells.     

Elevation of dCTP pools in xeroderma pigmentosum variant human fibroblasts alters the effects of DNA repair arrest by arabinofuranosyl cytosine

DNA excision repair inhibition by arabinofuranosyl cytosine (ara-C) or by ara-C/hydroxyurea (HU) was measured in log phase and confluent cultures of normal and xeroderma pigmentosium (XP)-variant human fibroblasts following insult by ultraviolet (UV) light (20 J/m²). Repair inhibition was determined by measuring the accumulation of DNA single-strand breaks/10⁸ daltons following cell culture exposure to ara-C or ara-C/HU in a series of 3 hr. pulses up ro 24 hr. after UV insult. Both normal and XP-variant derived cells showed a wide range of sensitivity to ara-C in log phase cells (0.2–9.4 breaks/10⁸ daltons DNA), although strand break accumulation was constant for each specific cell line. The same cells were more sensitive to ara-C/HU with a 2–14 fold increase in DNA strand breaks depending upon the cell line assayed. In confluent cultures of normal cells, maximum sensitivity to ara-C and ara-C/HU was achieved with similar levels of repair inhibition observed (16.1 and 16.5 breaks/10⁸ daltons, respectively). The same level of repair inhibition was observed in confulent XP-variants receiving ara-C/HU, but was reduced by 62–68% in cells treated with ara-C alone. Ara-C repair arrest was more rapidly reversed by competing concentrations of exogenous deoxycytidine (dCyd) in XP-variant compared to normal cells, especially in confluent cell cultures. In ara-C/HU treated cells, the level of dCyd reversal was reduced in the XP-variant when compared to cells exposed to ara-C alone. However, the same addition of HU had relatively little effect on dCyd reversal in normal cells. The measurements of dNTP levels indicate an elevated level of intracellular deoxycytosine triphosphate in XP-variant vs normal cells. The implications of these results are discussed as they relate to possible excision repair anomalies in the XP-variant.Abbreviations ara-C arabinofuranosul cytosine - dCTP deoxycytosine triphosphate - dCyd deoxycytidine - dNTP deoxynucleoside triphosphate - dT thymidine - HU hydroxyurea - XP xeroderma pigmentosium This research was sponsored jointly by the National Cancer Institute under Interagency Agreement #40-5-63, and the Office of Health and Environment Research, U. S. Department of Energy, under Contract W-7405-eng-26 with the Union Carbide Corporation.     

Azidocytidine is a specific inhibitor of deoxyribonucleotide synthesis in 3T6 cells

2'-Azidocytidine is a specific inhibitor of DNA synthesis in cultured 3T6 mouse fibroblasts. Earlier work (Akerblom, L., Pontis, E., and Reichard, P. (1982) 257, 6776-6782) indicated that the nucleoside, after phosphorylation, acted by inhibiting both ribonucleotide reduction and DNA strand elongation. We now demonstrate that the effect on strand elongation was due to a contamination of azidocytidine with less than 0.3% of arabinosyl cytosine. Pure azidocytidine inhibits specifically ribonucleotide reductase and its effects on DNA synthesis are secondary to this inhibition. The results with azidocytidine concerning the size and turnover of deoxyribonucleoside triphosphate pools parallel those of hydroxyurea and are distinct from those of arabinosyl cytosine. Together with hydroxyurea, azidocytidine is a useful compound in studies aiming at a specific block of the production of deoxynucleoside triphosphates in intact cells. Comparisons of the effects of azidocytidine and arabinosyl cytosine complement earlier studies with hydroxyurea and aphidicolin concerning inter-relations between dNTP synthesis and DNA replication.     

DNA repair synthesis in guinea pig pancreatic slices following in vitro exposure to N-nitrosomethylurethane.

The incorporation of thymidine into DNA in the presence of hydroxyurea (HU) by guinea pig pancreatic slices following exposure to N-nitrosomethylurethane (NMUT) was used to follow DNA repair synthesis. HU was used to suppress normal replicative DNA synthesis. Slices from the duodenal segment of the pancreas were exposed for periods of 15 to 90 min to NMUT at concentrations of 2 to 20 mM, then incubated in tritiated thymidine ([H3]-TdR) free of carcinogen, and radioactivity in DNA was determined. NMUT induced a a dose- and time-dependent increase in HU-insensitive thymidine incorporation. This stimulated incorporation, which could be attributed to repair synthesis, occurred immediately following the treatment and was largely complete within 3 h.     

dNTP pools determine fork progression and origin usage under replication stress

Intracellular deoxyribonucleoside triphosphate (dNTP) pools must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools are associated with increased mutagenesis, genomic instability and tumourigenesis. However, the mechanisms by which altered or imbalanced dNTP pools affect DNA synthesis remain poorly understood. Here, we show that changes in intracellular dNTP levels affect replication dynamics in budding yeast in different ways. Upregulation of the activity of ribonucleotide reductase (RNR) increases elongation, indicating that dNTP pools are limiting for normal DNA replication. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition to a slow-replication mode within minutes after S-phase entry. Upregulation of RNR activity delays this transition and modulates both fork speed and origin usage under replication stress. Interestingly, we also observed that chromosomal instability (CIN) mutants have increased dNTP pools and show enhanced DNA synthesis in the presence of HU. Since upregulation of RNR promotes fork progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.     

Replication of mammalian DNA in vitro. Evidence for initiation from fiber autoradiography

We have used fiber autoradiography to examine the DNA product made in vitro in a lysed cell system. CHO cells were treated with 0.01% Brij-58 and the lysates were incubated at 30 degrees C in a complete reaction mixture for in vitro DNA synthesis with [3H]thymidine triphosphate ([3H]TTP) as the radioactive tracer. Fiber autoradiograms prepared from the DNA showed that it was synthesized on tandemly arranged replication units that were of average size of 20 micrometers, very similar to the size of units found in vivo. The rate of replication fork movement was 25--50% of the in vivo rate. More than 80% of forks stopped functioning by 15 min, and 95% stopped by 60 min. This suggests that synthesis is halted by premature terminations. Evidence for new initiations was provided by replication units with labeled origins in DNA synthesized in an in vitro reaction in which radioactivity was omitted for the first 10 min of incubation. This, plus the observations that the distance between initiation points (replication unit size) is not increased and that premature termination accounts largely for the cessation of synthesis, suggest that significant initiation takes place in this in vitro replication system.     

Hydroxyurea arrests DNA replication by a mechanism that preserves basal dNTP pools

The relationship between dNTP levels and DNA synthesis was investigated using alpha factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G1/S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G1 levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37 degrees C before being released to 25 degrees C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G1 levels. Accumulation of dNTPs at G1/S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25 degrees C were able to replicate when subsequently switched to 37 degrees C in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold.     

Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity.

JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature sensitive for DNA replication (J. J. Dermody, B. E. Wojcik, H. Du, and H. L. Ozer, Mol. Cell. Biol. 6:4594-4601, 1986). It was chosen for detailed study because of its novel property of inhibiting both polyomavirus and adenovirus DNA synthesis in a temperature-dependent manner. Pulse-labeling studies demonstrated a defect in the rate of adenovirus DNA synthesis. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of uninfected cultures from 33 to 39 degrees C revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shift, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shift, in parallel with the decline in dNTP pool sizes. Moreover, the activity of cell extracts was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. The kinetics of dNTP pool size changes after temperature shift are quite distinct from those reported after inhibition of ribonucleotide reductase with hydroxyurea. An indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since human sequences other than those encoding the enzyme subunits can correct the temperature-sensitive growth defect in the mutant.     

Scintillometric determination of DNA repair in human cell lines: A critical appraisal

The ability of a variety of chemical and physical agents to stimulate DNA repair synthesis in human cell cultures was tested by a simplified scintillometric procedure, with the use of hydroxyurea (HU) to suppress DNA replicative synthesis. After incubation with [³H]thymidine, the radioactivity incorporated in to DNA was determined in controls (C) and treated (T) cultures and in the corresponding HU series (C_HU, T_HU). The ratios T_HU/C_HU and T_HU/T:C_HU/C, indicating absolute and relative increases of DNA radioactivity, were calculated. When both ratios were significantly higher than 1, they were taken as indices of DNA repair stimulation, whereas, no stimulation in inferred when both of them are ?1. The scintillometric estimate of DNA repair was always in agreement with the autoradiographic observations, so that the procedure adopted can be used as a rapid test for screening investigations.Agents giving a relative but no an absolute increase of DNA radioactivity are generally not inducers of repair synthesis as estimated by autoradiography. However, the same scintillometric results are also occasionally observed with DNA repair inducers, such as methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), owing to alterations of thymidine pool radioactivity. These chemicals, besides affecting the levels of labelled precursors in the intracellular pool in the T series, differently modified the increase of pool radioactivity which is a regular effect of HU. With such chemicals, DNA repair synthesis can be detected only after normalization of th DNA radioactivity on the basis of pool alterations.The quantitative value of the autoradiographic estimate of DNA repair is also affected by the changes in the radioactivity of the thymidine pool although autoradiography retains its qualitative value.Dimethylnitrosamine, mitomycin C and potassium dichromate, described by other authors as inducers of DNA repair, also gave negative results by the scintillometric procedure after normalization of DNA radioactivities. However, in our hands, these agents were unable to stimulated repair synthesis, according to the results of autoradiography and isopynic centrifugation.The proposed scintillometric procedure is effective in indicating false negative inducers of DNA repair, not giving rise to false positives.     

Deoxynucleoside triphosphate pool of mouse FM3A cell lines unaffected by mutagen treatment

The size of deoxynucleoside triphosphate pool in cultured mouse FM3A cells and mutator mutants isolated from this cell line was determined by high-pressure liquid chromatography after treatment of the cells with ultraviolet light, N-methyl-N′-nitro-N-nitrosoguanidine or mitomycin C. The results showed that, in all the FM3A cell clones, no large increase in the dATP or TTP pool was induced after treatment, while in some cases 40–50% decrease in dCTP pool was observed. It is concluded that the induction of large increase in dNTP pool is not the general effect of the mutagens.     

Deoxyribonucleoside triphosphate metabolism and the mammalian cell cycle. Effects of hydroxyurea on mutant and wild-type mouse S49 T-lymphoma cells

DNA precursor synthesis can be blocked specifically by the drug hydroxyurea (HU) which has therefore been used for anticancer therapy. High concentrations of HU, however, affect other processes than DNA synthesis; nevertheless, most studies on the biological action of HU have been made with concentrations at least one order of magnitude higher than those needed for cell-growth inhibition. In this study we characterized the effects of low concentrations of HU (i.e. concentrations leading to 50% inhibition of cell growth in 72 h) on cell cycle kinetics and nucleotide pools in mouse S49 cells with various defined alterations in DNA precursor synthesis. The effect of 50 microM HU on deoxyribonucleoside triphosphate pools was a 2-3-fold decrease in the dATP and dGTP pools, with no change in the dCTP pool and a certain increase in the dTTP pool. Addition of deoxycytidine or thymidine led to a partial reversal of the growth inhibition and cell-cycle perturbation caused by HU, and was accompanied by an increased level of the deoxyribonucleoside triphosphates. Addition of purine deoxyribonucleoside gave no protection, indicating that salvage of these nucleosides could not supply precursors for DNA synthesis in T-lymphoma cells. We observed a higher sensitivity to HU of cells lacking purine nucleoside phosphorylase or with a ribonucleotide reductase with altered allosteric regulation. Cells lacking thymidine kinase or deoxycytidine kinase were just as sensitive as wild-type cells.