Kinetics of incorporation of O6-methyldeoxyguanosine monophosphate during in vitro DNA synthesis

O6-Methyldeoxyguanosine triphosphate (m6dGTP), known to be produced in vivo by methylation of deoxyguanosine triphosphate with simple methylating mutagens, is utilized by prokaryotic DNA polymerases during in vitro replication of synthetic and natural DNA template-primers. A study of the kinetic behavior of m6dGTP during DNA replication in vitro and of its effect on DNA replication indicates that m6dGTP acts as an analogue of dATP with Kappm of about 6 microM for Escherichia coli DNA polymerase I (Klenow fragment) compared to the Kappm of about 0.8 microM for dATP. m6dGTP is not incorporated in the complete absence of dATP (a competitive inhibitor). m6dGTP also inhibits in vitro DNA synthesis. Different DNA polymerases behave differently in utilization and turnover of m6dGTP. T4 DNA polymerase shows stronger discrimination against m6dGMP incorporation than either T5 DNA polymerase or E. coli DNA polymerase I. The possibility that m6dGTP is unlikely to contribute significantly to in vivo mutation is discussed.     

Synchrony between DNA synthesis and thymidine incorporation into DNA in regenerating rat liver

DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz., thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation.The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz., thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone.It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed.     

In vivo labeling of fission yeast DNA with thymidine and thymidine analogs

In vivo labeling of DNA with thymidine and thymidine analogs has long been a cornerstone of replication studies. Unfortunately, yeast lack a thymidine salvage pathway and thus do not incorporate exogenous thymidine. Specifically, yeast neither efficiently take up exogenous thymidine from their growth media nor phosphorylate it to thymidylate, the precursor of dTTP. We have overcome these problems in fission yeast by expressing the human equilibrative nucleoside transporter 1 (hENT1) along with herpes simplex virus thymidine kinase (tk). hENT1 tk cells are healthy and efficiently incorporate exogenous thymidine and thymidine analogs. We present protocols for labeling DNA with tritiated thymidine, for in situ detection of incorporated BrdU by immunofluorescence, for double labeling with CldU and IdU, for CsCl gradient separation of IdU-labeled DNA, and for using hENT1 and tk as both positive and negative selection markers.     

1-beta-D-arabinofuranosylcytosine stimulates thymidine incorporation into the DNA of contact-inhibited cells

In rapidly proliferating cells l-β-d-arabinofuranosylcytosine (ara-C) is a potent inhibitor of DNA synthesis whose effect can be irreversible and consequently cytocidal. Whereas thymidine incorporation is greatly reduced in rapidly proliferating cells in the presence of ara-C, contact-inhibited cells, similarly treated, show increased thymidine incorporation by as much as 7-fold. This ara-C-induced stimulation appears to result from an influence on thymidine utilization rather than increased DNA synthesis.     

Osmotic stress inhibits thymidine incorporation into soybean protoplast DNA

DNA synthesis in protoplasts isolated from soybean cell suspension cultures has been investigated by [³H] thymidine uptake and incorporation kinetics. Initial rates of incorporation in exponential and 5-fluorodeoxyuridine synchronized protoplasts are inhibited by increased osmolarities of the medium. The inhibition was not readily reversible during 3 h culture in low osmotic medium. Velocity sedimentation analyses of replicating DNA from such protoplasts shows a complex pattern of inhibition. The inhibition probably effects replicon initiation as well as strand elongation and ligation of replication intermediates.     

Design and synthesis of boronic-acid-labeled thymidine triphosphate for incorporation into DNA

The boronic acid moiety is a versatile functional group useful in carbohydrate recognition, glycoprotein pull-down, inhibition of hydrolytic enzymes and boron neutron capture therapy. The incorporation of the boronic-acid group into DNA could lead to molecules of various biological functions. We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization. The synthesis was achieved using the Huisgen cycloaddition as the key reaction. We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA. DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection.     

Selective incorporation of thymidine in the organelle DNA of Polytoma obtusum

Polytoma obtusum was found to selectively incorporate exogenous thymidine into its leukoplast DNA. The nuclear DNA was unable to incorporate [3H]thymidine, although both DNA species could be labeled with radioactive adenine with similar efficiencies. The mitochondrial DNA (mitDNA), which had a buoyant density of 1.714 g/ml and was banded slightly on the heavier side of the nuclear DNA peak, was also found to incorporate a small amount of [3H]thymidine. These observations suggest that P. obtusum lack cytoplasmic thymidine kinase, whereas the enzyme is present in both leukoplast and mitochondria.     

Photoreversal-dependent release of thymidine and thymidine monophosphate from pyrimidine dimer-containing DNA excision fragments isolated from ultraviolet-damaged human fibroblasts

To elucidate the enzymatic excision-repair process operative on cyclobutane-type pyrimidine photodimers in human dermal fibroblasts, we have examined excised dimer-containing material recovered in the trichloroacetic acid soluble fraction from far-ultraviolet-irradiated (254 nm, 40 J m-2) and incubated (24 h) cell cultures. The excised DNA photoproducts were found in oligonucleotide fragments with an estimated mean chain length of approximately 3.7 bases. Exposure of these isolated excision fragments, labeled with [3H]thymidine (dT), to a secondary, dimer-photoreversing fluence of far-UV (5.5 kJ m-2) resulted in the release of free dT and thymidine monophosphate (TMP). Photorelease of these two radioactive species was measured by high-performance liquid chromatography, with TMP being detected as the increase in dT following bacterial alkaline phosphatase treatment. These data imply that the photoliberated dT and TMP moieties were attached to the excision fragments solely by the cyclobutane ring of the dimer. No evidence was obtained for the photoliberation of free thymine, thus corroborating a conclusion reached by others that the excision of dimers in human cells is not initiated by scission of an intradimer N-glycosyl bond. The sum of the tritium label recovered in dT plus TMP corresponded to approximately 40% of that disappearing from thymine-containing dimers on photoreversal, suggesting that in about 80% of the isolated excision fragments the dimer is located at one end of the oligonucleotide and contains a break in its internal phosphodiester bond.(ABSTRACT TRUNCATED AT 250 WORDS)     

Puromycin and cycloheximide inhibition of thymidine incorporation into DNA of cleaving sea urchin eggs

Puromycin has only a slight effect on the first period of thymidine incorporation (S1) in the fertilized Arbacia egg. Subsequent periods of incorporation are inhibited. Susceptibility of the second period of incorporation (S2) to puromycin is lost before and during S1.In late cleavage puromycin strongly inhibits phenylalanine incorporation into protein a few minutes in advance of its inhibition of thymidine incorporation into DNA. Thymidine incorporation is inhibited by 99 per cent after 65 min puromycin treatment. Cycloheximide inhibits both phenylalanine and thymidine incorporation by about 30 per cent in late cleavage. Preincubation of late-cleavage stages in thymidine greatly reduces the extent of inhibition of its incorporation by puromycin and cycloheximide, suggesting that thymidine may have been converted to other DNA precursors, and that the incorporation of these into DNA is less sensitive to protein synthesis inhibitors than are the initial steps in forming the precursors.The data suggest that the embryo must synthesize proteins in order to incorporate thymidine into DNA. In early stages these proteins may be synthesized about one duplication cycle in advance of their utilization.     

DNA repair and the genetic effects of thymidylate stress in yeast

Folate antagonists, such as aminopterin, methotrexate and various sulfonamides, block de novo thymidylate biosynthesis in Saccharomyces cerevisiae. The resulting starvation for thymine nucleotides is lethal and recombinagenic in RAD wild-type strains. In this paper we report our studies of these effects in repair-deficient yeast. Antifolate treatment of various rad mutants revealed that repair defects influence the killing and recombination caused by thymidylate deprivation. Compared to a RAD wild-type strain, diploids homozygous for rad3, rad6 or rad18 were more resistant to cell killing. Thus, contrary to findings with conventional DNA-damaging agents, the lethal effects of thymidylate starvation appear to be ameliorated by certain DNA repair deficiencies. On the other hand, a rad50 strain was extremely sensitive to the antifolates. Within this series of diploids, increasing sensitivity to thymidylate starvation was accompanied by an increase in recombination frequencies. The degrees of lethality and recombination, induced by thymidylate depletion, were correlated with the severity of DNA-strand breakage in the RAD and rad50 strains. Experiments with diploids homozygous for rad52, rad54 or rad57 suggested that aborted recombination events, provoked by thymidylate deprivation, caused chromosome loss. Furthermore, the repair defects in these mutants indicated that double-strand breaks are among the lethal lesions induced by thymine nucleotide starvation. Finally, we discuss the possibility that the recombinagenicity of thymidylate stress may account for one type of acquired resistance to methotrexate in mammalian cells.     

Synthesis and inhibitory activity of thymidine analogues targeting Mycobacterium tuberculosis thymidine monophosphate kinase

We report on Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt) inhibitory activities of a series of new 3′- and 5′-modified thymidine analogues including α- and β-derivatives. In addition, several analogues were synthesized in which the 4-oxygen was replaced by a more lipophilic sulfur atom to probe the influence of this modification on TMPKmt inhibitory activity. Several compounds showed an inhibitory potency in the low micromolar range, with the 5′-arylthiourea 4-thio-α-thymidine analogue being the most active one (K_i = 0.17 μM). This compound was capable of inhibiting mycobacteria growth at a concentration of 25 μg/mL.     

Measurement of bacterial growth rates in subsurface sediments using the incorporation of tritiated thymidine into DNA

Microbial growth rates in subsurface sediment from three sites were measured using incorporation of tritiated thymidine into DNA. Sampling sites included Lula, Oklahoma, Traverse City, Michigan, and Summit Lake, Wisconsin. Application of the thymidine method to subsurface sediments required (1) thymidine concentrations greater than 125 nM, (2) incubation periods of less than 4 hours, (3) addition of SDS and EDTA for optimum macromolecular extraction, and (4) DNA purification, in order to accurately measure the rate of thymidine incorporation into DNA. Macromolecule extraction recoveries, as well as the percentage of tritium label incorporated into the DNA fraction, were variable and largely dependent upon sediment composition. In general, sandy sediments yielded higher extraction recoveries and demonstrated a larger percentage of label incorporated into DNA than sediments that contained a high silt-clay component. Reported results also indicate that the acid-base hydrolysis procedure routinely used for macromolecular fractionation in water samples may not be routinely applicable to the modified sediment procedure where addition of SDS and EDTA are required for macromolecule extraction. Growth rates exhibited by subsurface communities are relatively slow, ranging from 5.1 to 10.2×10⁵ cells g^–1 day^–1. These rates are 2–1,000-fold lower than growth rates measured in surface sediments. These data lend support to the supposition that subsurface microbial communities are nutritionally stressed.     

The genetic control of dark recombination in yeast

The post-UV effects of dark holding and photoreactivating light on survival and intragenic recombination in UV-sensitive strains of yeast have been studied. The results indicate that two liquid holding dark repair pathways exist in this organism: the excision pathway which does not lead to intragenic recombination (“silent” repair) and a dark recombination pathway which does lead to intragenic recombination. A third pathway also exists which is plating-dependent and leads to intragenic recombination via a postreplication pathway.     

Age related changes in total DNA and RNA and incorporation of uridine and thymidine in rat brain

1. Total brain DNA and total brain RNA and the incorporation of thymidine[14C] and uridine[3H] were measured in young and aged rats. 2. From 20 days to the time of sexual maturation, both DNA and RNA levels increase. Total RNA exceeds total DNA at all ages. Comparatively, the ratio of total DNA/RNA is higher in young than in aged animals. 3. The incorporation of thymidine[14C]/g of DNA and of uridine[3H]/g of RNA decreases with age. This decrease is rapid in young animals. After 350 days of age, the incorporation becomes very low. The significance of data is discussed.     

Failure of tritiated thymidine incorporation into DNA to reflect the autoradiographically demonstrable calcium-induced increase in thymic lymphoblast DNA synthesis

Increasing the extracellular calcium concentration in thymic lymphocyte suspension from 0.6 to 1.8 mM stimulated the proliferation of the lymphoblast subpopulation as measured by increases in the proportion of cells autoradiographically labeled with ³H-TdR and in mitotic activity. However it was not possible to show this increased DNA synthesis by scintillometric measurement of the amount of ³H-TdR incorporated into extracted DNA. On the other hand, calcium did raise the incorporation of ¹⁴C-formate into the thymine residues of DNA, and increased the activity of isolated thymocyte thymidylate synthetase. In contrast to the mitogenic calcium ion, a thymidylate synthetase inhibitor, methotrexate, actually increased the incorporation of ³H-TdR into DNA. It is concluded that calcium increases the endogenous synthesis of thymidylate which in turn prevents the amount of incorporation of exogenous ³H-TdR from accurately reflecting the true level of DNA synthesis.