Chemical consequences of incorporation of 5-fluorouracil into DNA as studied by NMR

The cytotoxic analogue of thymine, 5-fluorouracil (Uf), is known to be incorporated into DNA in biological systems. This abnormal base has been synthetically incorporated into short DNA oligomers. The ionization of the N-3 proton of this base within DNA oligomers was measured by observation of the 19F chemical shift at varying pH values. The pKa values for the Uf ring of dTpdUfpdT and dApdUfpdA were determined to be 7.84 and 7.9, respectively. The self-complementary 12-mers d(G-C-G-C-A-A-T-Uf-G-C-G-C) and d(C-G-A-T-Uf-A-T-A-A-T-C-G) were synthesized, and 1H NMR was used to compare the helix dynamics and stability of the interstrand imino proton hydrogen bonds with those of the 12-mers d(G-C-G-C-A-A-T-T-G-C-G-C) and d(C-G-A-T-T-A-T-A-A-T-C-G). The N-3 hydrogen bond of the A-Uf base pair was less stable than the corresponding hydrogen bond in A-T base pairs in the same helix, and the A-Uf base pair was less stable than the A-T base pair in the analogous position of the control helix. The observed temperature-dependent dynamics and NMR melting temperatures of the control and dUf-containing oligomers were similar.     

A sensitive radiometric assay to measure D-xylulose kinase activity

A radiometric test system for D-xylulose kinase (XK) was developed for the measurement of enzyme activity in crude cell extracts and to minimize the volume of reaction mixtures besides increasing the sensitivity. [U-14C]xylulose 5-phosphate was produced from commercially available [U-14C]xylose in a coupled assay system containing D-xylose isomerase, which yields [U-14C]xylulose, the substrate of ATP-dependent D-xylulose kinase. Separation of products and substrates was achieved by thin layer chromatography, identification of radioactive spots by radioscanning followed by quantitative scintillation counting. The protocol was validated through determination of kinetic constants of a purified His-tagged enzyme from Escherichia coli and comparison with the spectrophotometric method. The radiometric assay was applied to determine xylulose kinase activity in crude cell extracts from a variety of eukaryotic and prokaryotic organisms.     

A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding

We present a simple, non-radioactive assay for DNA methyltransferase activity and DNA binding. As most proteins are studied as GFP fusions in living cells, we used a GFP binding nanobody coupled to agarose beads (GFP nanotrap) for rapid one-step purification. Immobilized GFP fusion proteins were subsequently incubated with different fluorescently labeled DNA substrates. The absolute amounts and molar ratios of GFP fusion proteins and bound DNA substrates were determined by fluorescence spectroscopy. In addition to specific DNA binding of GFP fusion proteins, the enzymatic activity of DNA methyltransferases can also be determined by using suicide DNA substrates. These substrates contain the mechanism-based inhibitor 5-aza-dC and lead to irreversible covalent complex formation. We obtained covalent complexes with mammalian DNA methyltransferase 1 (Dnmt1), which were resistant to competition with non-labeled canonical DNA substrates, allowing differentiation between methyltransferase activity and DNA binding. By comparison, the Dnmt1^C1229W catalytic site mutant showed DNA-binding activity, but no irreversible covalent complex formation. With this assay, we could also confirm the preference of Dnmt1 for hemimethylated CpG sequences. The rapid optical read-out in a multi-well format and the possibility to test several different substrates in direct competition allow rapid characterization of sequence-specific binding and enzymatic activity.     

A 32P postlabeling assay for determining the incorporation of bromodeoxyuridine into cellular DNA

Randerath's procedure for 32P postlabeling of 3'-monophosphate deoxyribonucleotides from digests of cellular DNA has been modified. 3'-Monophosphate deoxyribonucleotides are converted to 3',5'-bis[32P]phosphate deoxyribonucleotides with polynucleotide kinase and [32P]ATP; these products are enzymatically converted by P1 nuclease and polynucleotide kinase into 5'-[32P]monophosphate deoxyribonucleotides, which are separated from [32P]ATP on an anion-exchange column eluted with 0.1 M NaH2PO4, pH 6.5. Labeled mononucleotides in the effluent are separated by high-performance liquid chromatography. Values for the base composition of calf thymus DNA determined with this modified assay compare very favorably with reported values. The assay was used to measure the level of incorporation of the clinically useful agent bromodeoxyuridine into the DNA of 9L rat brain tumor cells. The modified assay appears to be a very accurate method for the determination of levels of base analogs incorporated into DNA.     

A sensitive fluorometric assay for the determination of DNA

A sensitive fluorometric assay for DNA determination using m-diaminobenzoic acid dihydrochloride for the reaction with deoxyribose liberated by perchloric acid is described. Fluorescence is proportional to the amount of DNA over the range 0.05–5 μg of DNA when the reaction is conducted in a volume of 400 μl and over the range 0.01–0.5 μg when the reaction is conducted in a volume of 40 μl. The assay is suitable for estimating DNA both in perchloric acid extracts and in complex mixtures with protein and RNA. The determination of DNA in cells grown as a monolayer by the described method is simple and rapid.     

A sensitive nonisotopic hybridization assay for HIV-1 DNA

We have developed a microtiter-based sandwich hybridization assay for the detection of low copy number HIV-1 sequences. The assay employs a capture DNA sequence covalently coupled to microtiter wells through linker arms. The detection probe is a biotin-labeled DNA fragment derived from sequences adjacent to the capture sequence. After hybridization in the presence of sample nucleic acid, the detection probe remains bound only if the sample contained complementary sequences spanning the junction between capture and detection probes. The amount of detection probe bound is quantified by incubation with a peroxidase-streptavidin conjugate and a colorimetric peroxidase substrate. This assay has been combined with enzymatic target amplification to achieve sensitive detection of HIV-1 in patient samples. Following amplification of HIV-1 DNA using the polymerase chain reaction technique, a 190-bp product is produced. This product is easily and specifically quantified using the sandwich hybridization assay. The resulting test can detect one HIV-1-infected cell in 10(5) cells or about 30 molecules of HIV-1 DNA.     

A simple and sensitive antibody-based method to measure UV-induced DNA damage inZea mays

We describe a western blotting procedure in which immobilized DNA is reacted with monoclonal antibodies specific to individual types of DNA damage. This method is applicable to small DNA samples and is more sensitive than the standard assay.     

A simple and sensitive DNA assay for plant extracts

A sensitive fluorimetric method was developed for the quantitative determination of DNA in plant (Zea mays L. and Medicago sativa L.) extracts. This method takes advantage of the specific increase in fluorescence intensity of the complex of DNA and the dye 4′,6′-diamidino-2-phenylindole (DAPI). Recovery of DNA and dissociation of histones from DNA were maximized by the addition of 2.0 molar NaCl to the homogenates. Treatment of the homogenate with chloroform to remove pigments and proteins decreased the quenching of fluorescence of the DAPI-DNA complex. The fluorescence intensity of RNA with DAPI was less than 2% of that produced by an equivalent weight of DNA. Comparisons were made between this fluorimetric DNA method and the commonly used diphenylamine assay for DNA. The diphenylamine DNA assay was more timeconsuming, less sensitive, and consistently resulted in lower estimates of DNA concentrations than did the fluorimetric DNA assay.     

UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation

Cytotoxicity of 5-fluorouracil (FU) and 5-fluoro-2′-deoxyuridine (FdUrd) due to DNA fragmentation during DNA repair has been proposed as an alternative to effects from thymidylate synthase (TS) inhibition or RNA incorporation. The goal of the present study was to investigate the relative contribution of the proposed mechanisms for cytotoxicity of 5-fluoropyrimidines. We demonstrate that in human cancer cells, base excision repair (BER) initiated by the uracil–DNA glycosylase UNG is the major route for FU–DNA repair in vitro and in vivo. SMUG1, TDG and MBD4 contributed modestly in vitro and not detectably in vivo. Contribution from mismatch repair was limited to FU:G contexts at best. Surprisingly, knockdown of individual uracil–DNA glycosylases or MSH2 did not affect sensitivity to FU or FdUrd. Inhibitors of common steps of BER or DNA damage signalling affected sensitivity to FdUrd and HmdUrd, but not to FU. In support of predominantly RNA-mediated cytotoxicity, FU-treated cells accumulated ~3000- to 15 000-fold more FU in RNA than in DNA. Moreover, FU-cytotoxicity was partially reversed by ribonucleosides, but not deoxyribonucleosides and FU displayed modest TS-inhibition compared to FdUrd. In conclusion, UNG-initiated BER is the major route for FU–DNA repair, but cytotoxicity of FU is predominantly RNA-mediated, while DNA-mediated effects are limited to FdUrd.     

A non-radioactive, PFGE-based assay for low levels of DNA double-strand breaks in mammalian cells

A PFGE method was adapted to measure DNA double-strand breaks (DSBs) in mammalian cells after low (0-25 Gy) doses of ionising radiation. Instead of radionuclide incorporation, DNA staining in the gel by SYBR-Gold was used, which lowered the background of DNA damage and could be applied to non-cycling cells. DSB level was defined as a product of a fraction of DNA released to the gel (FR) and a number of DNA fragments in the gel (DNA(fragm)) and expressed as a percentage above control value. The slope of the dose-response curve was two-fold higher compared to that with FR alone as DSB level indicator (31.4 versus 15.6% per Gy). Two alternative ways were proposed to determine the total amount of DNA, used for FR calculation: measurement of DNA content in a plug not subjected to electrophoresis, with the use of Pico-Green, or estimation of DNA released to the gel from a plug irradiated with 600 Gy of gamma-rays. The limit of DSB detection was 0.25 Gy for human G1-lymphocytes and 0.5-1 Gy for asynchronous cultures of human glioma M059 K and J or mouse lymphoma L5178Y-R and -S cells. Specificity of our PFGE assay to DSB was confirmed by the fact that no damage was detected after treatment of the cells with H(2)O(2), an inducer of single-strand DNA breaks (SSBs). On the contrary, the H(2)O(2) inflicted damage was detected by neutral comet assay, attaining 160% above control (equivalent to 2.5 Gy of X-radiation). DSB rejoining, measured in cells after X-irradiation with a dose of 10 Gy, generally proceeded faster than that measured previously after higher (30-50 Gy) doses of ionising radiation. Clearly seen were defects in DSB rejoining in radiosensitive M059 J and L5178Y-S cells compared to their radioresistant counterparts, M059 K and L5178Y-R. In some cell lines, a secondary post-irradiation increase in DSB levels was observed. The possibility is considered that these additional DSBs may accumulate during processing of non-DSB clustered DNA damage or/and represent early apoptotic events.     

In vivo 19F-NMR of 5-fluorouracil incorporation into RNA and metabolites in Escherichia coli cells

19F resonances from RNA with 5-fluorouracil incorporated could be observed in intact Escherichia coli cells, as well as in tRNA isolated from the cells. 19F-NMR signals from the metabolic breakdown products of the fluorinated RNA were also detected in vivo. By observing the 19F-NMR spectrum, variations in the metabolic disposition of administered 5-fluorouracil could be monitored as a function of time and be compared when the cells were deprived of oxygen and other nutrients, subjected to ethidium bromide treatment, or grown in the presence of mitomycin C.     

In vivo sister-chromatid exchange: A sensitive measure of DNA damage

A variety of chemical agents and X-irradiation were examined for their abilities to induced sister-chromatid exchanges (SCE) in vivo. In addition to demonstrating the several known mutagens and carcinogens are capable of inducing SCE in vivo, our studies indicate that the suspected carcinogen, tris-bromophosphate, can significantly elevate SCE levels. Comparison of the effects of these agents on SCE levels, chromosomal-aberration frequencies and cell-replication kinetics reveals that no consistent relationship exists between SCE levels and other indicators of cellular DNA damage.

It is proposed that analysis of SCE induction in vivo may provide a useful technique for the screening of mutagenic and carcinogenic compounds.