Deoxyribonucleotide Triphosphates Research Articles

The effectiveness of modified sperm washes in severely oligoasthenozoospermic men infected with human immunodeficiency and hepatitis C viruses

Men who are seropositive for human immunodeficiency virus (HIV) or hepatitis C virus now can father children safely, thanks to sperm washing and nested polymerase chain-reaction techniques, followed by intracytoplasmic sperm injection. However, in a small percentage of such patients, it is impossible to recover spermatozoa after said procedures because of their highly impaired spermatogenesis. We have established that less rigorous methods, such as repeated centrifugation, yield nested polymerase chain-reaction HIV- and hepatitis C virus-negative specimens, even in sperm samples from men with severe oligoasthenozoospermia.

C4′ sugar oxidation of deoxyribonucleotide triphosphates by chromium(V) complexes

The Cr(V) complexes, bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) ([OCr V(ehba) 2] −) and (2,2-bis(hydroxymethyl)-2-(bis(2-hydroxyethyl)amino)ethanolato)oxochromate(V) ([OCr V(BT)] 2−), were reacted with a series of deoxyribonucleotide triphosphates. Oxidation of deoxyribose at C4′ was observed by measuring the amount of thiobarbituric acid reactive species (TBARS) produced in these reactions. For both compounds, the TBARS obtained with purine nucleotides was between 2.25 and 3.5 times greater than what was observed with pyrimidine nucleotide. This result suggests that the identity of the nucleic acid base can influence the hydrogen atom abstraction at C4′. Overall, the amount of product obtained with [OCr V(BT)] 2− was significantly less than what was observed with [OCr V(ehba) 2] −, indicating that these two Cr(V) model complexes may oxidize DNA differently.

Genome-wide replication profiles of S-phase checkpoint mutants reveal fragile sites in yeast

The S-phase checkpoint kinases Mec1 and Rad53 in the budding yeast, Saccharomyces cerevisiae, are activated in response to replication stress that induces replication fork arrest. In the absence of a functional S-phase checkpoint, stalled replication forks collapse and give rise to chromosome breakage. In an attempt to better understand replication dynamics in S-phase checkpoint mutants, we developed a replication origin array for budding yeast that contains 424 of 432 previously identified potential origin regions. As expected, mec1-1 and rad53-1 mutants failed to inhibit late origin activation. Surprisingly however, 17 early-firing regions were not replicated efficiently in these mutants. This was not due to a lack of initiation, but rather to problems during elongation, as replication forks arrested in close proximity to these origins, resulting in the accumulation of small replication intermediates and eventual replication fork collapse. Importantly, these regions were not only prone to chromosome breakage in the presence of exogenous stress but also in its absence, similar to fragile sites in the human genome.

Clinical Spectrum of Mitochondrial DNA Depletion Due to Mutations in the Thymidine Kinase 2 Gene

Mitochondrial DNA depletion syndrome is an autosomal recessive disorder characterized by decreased mitochondrial DNA copy numbers in affected tissues. It has been linked to 4 genes involved in deoxyribonucleotide triphosphate metabolism: thymidine kinase 2 (TK2), deoxyguanosine kinase (DGUOK), polymerase gamma (POLG), and SUCLA2, the gene encoding the beta-subunit of the adenosine diphosphate-forming succinyl coenzyme A synthetase ligase. To highlight the variability in the clinical spectrum of TK2-related mitochondrial DNA depletion syndrome. Review of patients and the literature. Tertiary care university. Four patients with mitochondrial DNA depletion syndrome and mutations in the TK2 gene. Definition of clinical variability. Patient 1 had evidence of lower motoneuron disease and was initially diagnosed as having spinal muscular atrophy type 3. Patient 2, who is alive and ambulatory at age 9 years, presented at age 2 years with a slowly progressive mitochondrial myopathy. Patient 3 had a more severe myopathy, with onset in infancy and death at age 6 years of respiratory failure. Patient 4 had a rapidly progressive congenital myopathy with rigid spine syndrome and he died at age 19 months. The clinical spectrum of TK2 mutations is not limited to severe infantile myopathy with motor regression and early death but includes spinal muscular atrophy type 3-like presentation, rigid spine syndrome, and subacute myopathy without motor regression and with longer survival.

Significant enhancement of fluorescence on hybridization of a molecular beacon to a target DNA in the presence of a site-specific DNA nickase

We have developed a simple isothermal (55 °C) reaction that permits detection of DNA targets using only two components: a molecular beacon and a site-specific DNA nickase without deoxyribonucleotide triphosphates and primers. The loop sequence of the molecular beacon should contain a DNA nickase recognition site. The nickase–molecular beacon (NMB) combination permits a 100-fold increase in fluorescent signal. The applications of the NMB assay for enhancement of fluorescent signal in some isothermal methods are discussed.

Identification of Three gp350/220 Regions Involved in Epstein-Barr Virus Invasion of Host Cells

Epstein-Barr virus (EBV) invasion of B-lymphocytes involves EBV gp350/220 binding to B-lymphocyte CR2. The anti-gp350 monoclonal antibody (mAb)-72A1 Fab inhibits this binding and therefore blocks EBV invasion of target cells. However, gp350/220 regions interacting with mAb 72A1 and involved in EBV invasion of target cells have not yet been identified. This work reports three gp350/220 regions, defined by peptide 11382, 11389, and 11416 sequences, that are involved in EBV binding to B-lymphocytes. Peptides 11382, 11389, and 11416 bound to CR2(+) but not to CR2(-) cells, inhibited EBV invasion of cord blood lymphocytes (CBLs), were recognized by mAb 72A1, and inhibited mAb 72A1 binding to EBV. Peptides 11382 and 11416 binding to peripheral blood lymphocytes (PBLs) induced interleukin-6 protein synthesis in these cells, this phenomenon being inhibited by mAb 72A1. The same behavior has been reported for gp350/220 binding to PBLs. Anti-peptide 11382, 11389, and 11416 antibodies inhibited EBV binding and EBV invasion of PBLs and CBLs. Peptide 11382, 11389, and 11416 sequences presented homology with the C3dg regions coming into contact with CR2 (C3dg and gp350 bound to similar CR2 regions). These peptides could be used in designing strategies against EBV infection.

Neurochondrin Negatively Regulates CaMKII Phosphorylation, and Nervous System-specific Gene Disruption Results in Epileptic Seizure

Neurochondrin is a novel cytoplasmic protein and possibly involved in neurite outgrowth, chondrocyte differentiation, and bone metabolism. Our previous trial in disclosing its role by the loss of function in mice failed because of the lethality in utero. In this study, we eliminated the neurochondrin gene expression preferentially in the nervous system by the conditional knockout strategy. Our results showed that neurochondrin is a negative regulator of Ca(2+)/calmodulin-dependent protein kinase II phosphorylation and essential for the spatial learning process but not for the differentiation or neurite outgrowth of the neuron. In addition, the nervous system-specific homozygous gene disruption resulted in epileptic seizure.

A Degrading Job

Checkpoint responses lead to cell cycle arrest following DNA damage, which allows time for DNA repair. In Saccharomyces cerevisiae , Rad53 kinase is critical to this arrest and also for deoxyribonucleotide triphosphate production during S phase. Most of Rad53's functions depend on its activation by the upstream checkpoint kinase Mec1; however, rad53 deletion mutants show a slow-growth phenotype, whereas mec1 deletion mutants do not. During S phase, histone synthesis is coordinated with DNA replication, and newly synthesized histones are deposited behind the replication fork by chaperones (see Quivy and Almouzni). Gunjan and Verreault investigated the role of Rad3, which can interact with the histone chaperone Asf1, in histone metabolism in wild-type and mutant strains of S. cerevisiae . Yeast lacking--or expressing kinase-deficient--Rad3 were hypersensitive to histone overexpression (which is toxic), whereas yeast lacking Mec1 were not. Western analysis of tagged overexpressed histones or of fractionated or chaperone-bound endogenous histones indicated that degradation of excess histones that were not packaged into chromatin was impaired in rad53 mutants. Therefore, rad53 mutant cells accumulated excess histones. Histone overexpression increased sensitivity to DNA damage, without directly damaging DNA, and reducing histone gene dosage suppressed the rad53 mutant phenotype of sensitivity to DNA damage, slow growth, and chromosome loss. Rad53 formed a complex with histones that did not include Asf1 or any other known histone chaperone proteins; complex formation was affected by Rad53 kinase activity. Thus, Rad53 appears to have a novel function of monitoring and maintaining histone levels that is independent of Mec1 and its activation by DNA damage. A. Gunjan, A. Verreault, A Rad53 kinase-dependent surveillance mechanism that regulates histone protein levels in S. cerevisiae . Cell 115 , 537-549 (2003). [Online Journal] J.-P. Quivy, G. Almouzni, Rad53: A controller ensuring the fine-tuning of histone levels. Cell 115 , 508-510 (2003). [Online Journal]

Tight binding of deoxyribonucleotide triphosphates to human thymidine kinase 2 expressed in Escherichia coli. Purification and partial characterization of its dimeric and tetrameric forms.

Human thymidine kinase 2 (hTK2) phosphorylates pyrimidine deoxyribonucleosides to the corresponding nucleoside monophosphates, using a nucleotide triphosphate as a phosphate donor. In this study, hTK2 was cloned and expressed at high levels in Escherichia coli as a fusion protein with maltose-binding protein. Induction of a heat-shock response by ethanol and coexpression of plasmid-encoded GroEL/ES chaperonins at 28 degrees C minimized the nonspecific aggregation of the hybrid protein and improved the recovery of three homooligomeric forms of the properly folded enzyme, i.e., dimer > tetramer > hexamer. The dimer and the tetramer were isolated in stable and highly purified forms after proteolytic removal of the fusion partner. Both oligomers contained a substoichiometric amount of deoxyribonucleotide triphosphates (dTTP > dCTP > dATP), known to be strong feedback inhibitors of the enzyme. Steady-state kinetic studies were consistent with the presence of endogenous inhibitors, and both oligomeric forms revealed a lag phase of at least approximately 5 min, which was abolished on preincubation with substrate (dThd or dCyd). The rather similar kinetic properties of the two oligomeric forms indicate that the basic functional unit is a dimer. Molecular docking experiments with a modeled hTK2 three-dimensional structure accurately predicted the binding positions at the active site of the natural substrates (dThd, dCyd, and ATP) and inhibitors (dTTP and dCTP), with highly conserved orientations obtained for all ligands. The calculated relative nonbonded interaction energies are in agreement with the biochemical data and show that the inhibitor complexes have lower stabilization energies (higher affinity) than the substrates.

Characterization of the DNA Damage-inducible Helicase DinG from Escherichia coli

The dinG promoter was first isolated in a genetic screen scoring for damage-inducible loci in Escherichia coli (Lewis, L. K., Jenkins, M. E., and Mount, D. W. (1992) J. Bacteriol. 174, 3377-3385). Sequence analysis suggests that the dinG gene encodes a putative helicase related to a group of eukaryotic helicases that includes mammalian XPD (Koonin, E. V. (1993) Nucleic Acids Res. 21, 1497), an enzyme involved in transcription-coupled nucleotide excision repair and basal transcription. We have characterized the dinG gene product from E. coli using genetic and biochemical approaches. Deletion of dinG has no severe phenotype, indicating that it is non-essential for cell viability. Both dinG deletion and over-expression of the DinG protein from a multicopy plasmid result in a slight reduction of UV resistance. DinG, purified as a fusion protein from E. coli cells, behaves as a monomer in solution, as judged from gel filtration experiments. DinG is an ATP-hydrolyzing enzyme; single-stranded (ss) DNA stimulates the ATPase activity 15-fold. Kinetic data yield a Hill coefficient of 1, consistent with one ATP-hydrolyzing site per DinG molecule. DinG possesses a DNA helicase activity; it translocates along ssDNA in a 5' --> 3' direction, as revealed in experiments with substrates containing non-natural 5'-5' and 3'-3' linkages. The ATP-dependent DNA helicase activity of DinG requires divalent cations (Mg2+, Ca2+, and Mn2+) but is not observed in the presence of Zn2+. The DinG helicase does not discriminate between ribonucleotide and deoxyribonucleotide triphosphates, and it unwinds duplex DNA with similar efficiency in the presence of ATP or dATP. We discuss the possible involvement of the DinG helicase in DNA replication and repair processes.

Nevirapine-selected mutations Y181I/C of HIV-1 reverse transcriptase confer cross-resistance to stavudine.

Stavudine administration did not increase the frequency of Y181I/C reverse transcriptase (RT) mutations in non-nucleoside reverse transcriptase inhibitor (NNRTI)-treated patients. However, recombinant Y181C HIV-1 showed reduced stavudine susceptibility with respect to both recombinant wild-type and K103N HIV-1 strains. In addition, recombinant Y181I RT enzyme showed reduced susceptibility to stavudine with respect to both wild-type and K103N RT. A previously unnoticed role of Y181I/C RT changes selected by nevirapine or other NNRTI in determining stavudine resistance is documented.

Overexpression of the R2 subunit of ribonucleotide reductase in human nasopharyngeal cancer cells reduces radiosensitivity.

Ribonucleotide reductase is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleotide triphosphates, which are utilized in both DNA synthesis and DNA repair. We reported previously that RR enzyme activity and R2 (catalytic subunit of RR) protein levels were increased after exposure to ionizing radiation (IR) in growth-arrested human tumor cells, suggesting that R2 protein expression regulates RR activity to allow for IR damage repair. Using isogenic human nasopharyngeal carcinoma cells in this study, we examine the relationship of overexpression of either the R1 regulatory subunit or the R2 catalytic subunit of RR to the cellular response of IR damage. We used three isogenic human nasopharyngeal cancer cell lines previously derived by Zhou et al, including KB, the parental tumor cell line; KB/M1, an R1 protein-overexpressing clone stably transfected with human R1 complementary DNA; and KB/M2, a R2 protein-overexpressing clone stably transfected with human R2 complementary DNA. We initially characterized these isogenic human tumor cell lines in exponential growth for R2 protein expression, RR enzyme activity, and R2 protein changes during the cell cycle by flow cytometry. Subsequently, the IR response in these cell lines was determined by clonogenic survival, cell cycle changes occurring after IR, and an analysis of IR DNA damage determined by pulsed field gel electrophoresis. The effect of combining IR and hydroxyurea, a RR (R2) inhibitor, was also studied in KB and KB/M2 cells. KB/M2 cells were found to have 4.5-fold higher R2 protein expression and a threefold higher RR enzyme activity in exponential growth than KB and KB/M1. Although R2 protein levels increased at the G1/S transition in all cell lines, KB/M2 cells also demonstrated consistently higher R2 protein levels throughout the cell cycle. Using a linear-quadratic analysis of IR clonogenic survival data, KB/M2 cells were more radioresistant than KB and KB/M1 cells, including both decreased alpha and decreased beta values, a finding that correlates with increased reparable IR damage. KB/M2 cells also show a reduced G2 cell cycle arrest and fewer DNA double strand breaks 18 hours after IR (6 Gy). Exposure of KB/M2 cells to hydroxyurea (300 microM) after exposure to IR restored in vitro radiosensitivity in a manner similar to that found in KB and KB/M1 cells. An increase in R2 protein levels and RR activity in KB/M2 cells results in IR resistance, which appears mediated by enhanced IR damage repair during G2. R1 protein overexpression in these isogenic human tumor cells (KB/M1) did not affect RR activity or IR response.

Repair system for noncanonical purines in Escherichia coli.

Exposure of Escherichia coli strains deficient in molybdopterin biosynthesis (moa) to the purine base N-6-hydroxylaminopurine (HAP) is mutagenic and toxic. We show that moa mutants exposed to HAP also exhibit elevated mutagenesis, a hyperrecombination phenotype, and increased SOS induction. The E. coli rdgB gene encodes a protein homologous to a deoxyribonucleotide triphosphate pyrophosphatase from Methanococcus jannaschii that shows a preference for purine base analogs. moa rdgB mutants are extremely sensitive to killing by HAP and exhibit increased mutagenesis, recombination, and SOS induction upon HAP exposure. Disruption of the endonuclease V gene, nfi, rescues the HAP sensitivity displayed by moa and moa rdgB mutants and reduces the level of recombination and SOS induction, but it increases the level of mutagenesis. Our results suggest that endonuclease V incision of DNA containing HAP leads to increased recombination and SOS induction and even cell death. Double-strand break repair mutants display an increase in HAP sensitivity, which can be reversed by an nfi mutation. This suggests that cell killing may result from an increase in double-strand breaks generated when replication forks encounter endonuclease V-nicked DNA. We propose a pathway for the removal of HAP from purine pools, from deoxynucleotide triphosphate pools, and from DNA, and we suggest a general model for excluding purine base analogs from DNA. The system for HAP removal consists of a molybdoenzyme, thought to detoxify HAP, a deoxyribonucleotide triphosphate pyrophosphatase that removes noncanonical deoxyribonucleotide triphosphates from replication precursor pools, and an endonuclease that initiates the removal of HAP from DNA.

Liquid chromatography–tandem mass spectrometry assays for intracellular deoxyribonucleotide triphosphate competitors of nucleoside antiretrovirals

This study was aimed to apply an LC–MS–MS method previously developed for intracellular nucleoside reverse transcriptase inhibitors-triphosphate (NRTI-TPs) to the determination of natural deoxyribonucleotides (dNTPs) in human peripheral blood mononuclear cells. The LC–MS–MS method was directly used in assay of dATP and dTTP. Interferences by ribonucleotides (rNTPs) prevented direct application to the two other analytes: dGTP and dCTP. A periodate oxidation procedure was therefore optimized to remove rNTPs from the cell medium in order to quantitate dCTP and dGTP. The determination of the intracellular ratio of NRTI-TP/dNTP in HIV-infected patients now involves use of the same chromatographic system for simultaneous assay of several analytes.

Implications of hydrophobicity and free energy of solvation for characterization of nucleic acids by electrospray ionization mass spectrometry.

Electrospray ionization (ESI) is a dynamic process that, when coupled with mass spectrometry (MS), serves as an invaluable tool for analysis of biomolecules. Our group, as well as others, has observed that there is a bias in signal intensity for one strand of a PCR amplicon over the complementary strand in an ESI mass spectrum. In this report, we have investigated the contributions of hydrophobicity and free energy of solvation to relative signal intensities in ESI-MS spectra of nucleic acids. We developed approaches for predicting which strand of the PCR amplicon will be the most intense: one based on a rate equation for calculating ion flux using values from the literature for hydrophobicity and free energy of solvation and the other based on the percentage of the relatively hydrophilic guanines present in the strand. A trend in signal intensity for deoxyribonucleotide triphosphates, oligonucleotides, and PCR amplicons was observed that was consistent with our model. On the basis of the observation that increased hydrophobicity correlates with greater signal intensity, we selectively enhanced the signal intensity of a 20-mer with the addition of an alkyl chain to the 5' terminus, which subsequently improved the limit of detection to 1 nM, an improvement by 1 order of magnitude. This was extended to a 53-bp PCR amplicon by modifying one primer with the hydrophobic moiety, which resulted in a 16% increase in signal intensity. We capitalized on this result to determine allele frequencies from pooled DNA for single-nucleotide polymorphisms down to 1%.

Ribonucleotide reductase and thymidine phosphorylation: two potential targets of azodicarbonamide

Azodicarbonamide tested as an anti-HIV agent was reported to expulse zinc from viral zinc-cysteine factors and to inhibit calcium mobilization machinery. It has structural analogy with hydroxyurea that inhibits ribonucleotide reductase and could also act on this target. Azodicarbonamide was therefore tested for its capacity to modulate deoxyribonucleotides triphosphate pools alone or in combination with other agents in the lymphoblastic SUP-T1 cell line susceptible to HIV infection. The deoxyribonucleotides triphosphate were evaluated by an enzymatic assay using sequenase. Two hours exposure of SUP-T1 cells to 100 μM azodicarbonamide induced a 50% reduction of each deoxyribonucleotide triphosphate. Among other inhibitors of nucleotide metabolism (hydroxyurea, methotrexate and thymidine), hydroxyurea only reproduces the effect of azodicarbonamide. This suggests, but does not demonstrate directly, that azodicarbonamide inhibits ribonucleotide reductase activity. The combination of azodicarbonamide with each of these inhibitors affected particularly the dCTP pool. During this study it was also suggested that azodicarbonamide could interfere with thymidine phosphorylation. Thymidine phosphorylating activity was measured with 3H-thymidine as substrate. In acellular preparations, azodicarbonamide also non-competitively inhibits thymidine phosphorylating activity. This effect was not reproduced by hydroxyurea. Thus, in vitro azodicarbonamide decreases the intracellular pool of deoxyribonucleotide and thymidine phosphorylation.

Human DNA polymerase beta initiates DNA synthesis during long-patch repair of reduced AP sites in DNA.

Simple base damages are repaired through a short-patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase beta (Pol beta) is responsible for the repair synthesis in this pathway and also removes a 5'-sugar phosphate residue by catalyzing a beta-elimination reaction. How ever, some DNA lesions that render deoxyribose resistant to beta-elimination are removed through a long-patch repair pathway that involves strand displacement synthesis and removal of the generated flap by specific endonuclease. Three human DNA polymerases (Pol beta, Pol delta and Pol epsilon) have been proposed to play a role in this pathway, however the identity of the polymerase involved and the polymerase selection mechanism are not clear. In repair reactions catalyzed by cell extracts we have used a substrate containing a reduced apurinic/apyrimidinic (AP) site resistant to beta-elimination and inhibitors that selectively affect different DNA polymerases. Using this approach we find that in human cell extracts Pol beta is the major DNA polymerase incorporating the first nucleotide during repair of reduced AP sites, thus initiating long-patch base excision repair synthesis.

Toward a biologically based dose-response model for developmental toxicity of 5-fluorouracil in the rat: a mathematical construct.

Biologically based dose-response (BBDR) models comprise one way to incorporate mechanistic information into a dose-response assessment to be used for risk assessments. The chemotherapeutic drug 5-fluorouracil (5-FU) has been used as a prototypic compound for the construction of a BBDR model for developmental toxicity. Previous work has provided data and a general mechanistic framework for the developmental toxicity of 5-FU when it was administered to pregnant rats subcutaneously on gestation day 14. In this paper, a mathematical model relating maternally administered treatment with 5-FU to embryonal thymidylate synthetase inhibition and thymidylate synthetase inhibition to various measures of deoxyribonucleotide triphosphate (dNTP) pool perturbation is developed, and parameters are estimated using the data collected. The strategy used was to develop semi-empirical submodels for each of the intervening steps, and to estimate model parameters from previously described data. The models developed predict that there is no practical threshold for dNTP pool perturbation; that is, even minimal doses of 5-FU should result in some perturbation of dNTP pools. In particular, the relationship between dNTP pool perturbation and fetal weight deficit suggests that if there is a biological threshold for the effect of 5-FU on fetal weight, the responsible repair or compensation mechanism must be downstream of dNTP pool perturbation, and saturable at 5-FU doses lower than 10 mg/kg (the lowest dose examined for developmental effects in these studies).

Inhibition of ribozymes by deoxyribonucleotides and the origin of DNA.

Two catalytic functions were required, minimally, for the appearance of DNA in evolution: a ribonucleotide reductase (RNR) and a reverse transcriptase (RT). If one accepts the explanatory strength of the RNA world model, it is clear that DNA molecules arose in the RNA world at some stage during the early evolution of cells. I suggest that competition for limited and valuable resources such as nucleotides, amino acids, and sugars made an early appearance among RNA cells, RNA viruses, viroids, and RNA plasmids. Structural and functional similarities between the different types of polymerases favor the simple hypothesis that the first RTs were RNA polymerase mutants that preferentially joined together preexisting deoxyribonucleotide triphosphates (dNTPs) using RNA templates. What was the role of dNTPs inside cells before DNA was synthesized and tested by natural selection? The oxygen atom that is removed by the reductase is of crucial importance to many ribozyme functions, since the 2'-OH is a strong nucleophile that forms transitional states during catalysis. Consequently, a RNR may have been used by cellular parasites to inhibit ribozyme action. Thus, DNA may have been, initially, an inert by-product of retrotranscription in lineages that acquired RTs and could synthesize DNA molecules using cellular RNA templates to detoxify the intracellular environment. DNA was useless as template until a transcriptase (DNA-dependent RNA polymerase) evolved that could copy (-)DNA to reconstitute the (+)RNA genome, indeed a successful way of confronting ribonuclease threats in the RNA world.

Comparison of cell proliferation following partial hepatectomy in rats fed NIH-31 or semipurified AIN-76A diets: Effects of nucleic acid supplementation

Male Fischer 344 rats, were administered NIH-31, AIN-76A, or AIN-76A plus 0.25% yeast RNA diets for 3 weeks, partially hepatectomized (PH), and groups of 6 rats from each diet group were sacrificed at 0, 2, 7 and 14 days following PH. Liver weight, total DNA, total lipids, levels of thiobarbaturic acid reactive substances (TBARS), and levels of intracellular deoxyribonucleotides were determined. In addition, the number of hepatocytes in mitosis and/or positive for nuclear proliferating cell nuclear antigen (PCNA) were determined. Liver regeneration, measured as liver weight or total liver DNA, was equal in the three diet groups and was essentially complete by the end of 14 days. However, there was a 53% reduction in the mitotic index and a two-fold increase in the ratio of proliferating cells (G1 + S + G2 cells) to mitoses at 48 hours in animals of the AIN-76A diet group compared to those fed NIH-31 diet, suggesting that the cells were arrested or delayed in S-phase. At the time of PH, liver deoxyuridine monophosphate (dUMP) and deoxythymidine triphosphate (dTTP) levels in the AIN-76A group were decreased 29% and 57%, respectively, compared to those of the NIH-31 group. Addition of yeast RNA to the AIN-76A diet partially prevented the decrease in mitosis and maintained deoxyribonucleotide triphosphates (dNTP) levels to those of the NIH-31 group. These results indicate that the lack of dietary nucleotides in semipurified diets can alter dNTP levels and have adverse effects on cell proliferation. Consideration should be given to supplementing purified diets with a dietary source of nucletides.