Alkaline Sucrose Density Gradient Sedimentation Research Articles

A large intermediate domain of vertebrate REV3 protein is dispensable for ultraviolet-induced translesion replication

DNA polymerase ζ (pol ζ) is involved in translesion replication (translesion synthesis, TLS) and plays an essential role in embryogenesis. In adults, pol ζ triggers mutation as a result of error-prone TLS and causes carcinogenesis. The catalytic subunit of pol ζ, REV3, is evolutionarily conserved from yeast and plants to higher eukaryotes. However, the structures are notably different: unlike that in yeast REV3, a large intermediate domain is inserted in REV3 of humans and mice. The domain is mostly occupied with noncommittal structures (random coil…etc.); therefore, its role and function are yet to be resolved. Previously, we reported deficient levels of ultraviolet (UV)-induced TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). Here, we constructed a mouse Rev3-expressing plasmid with a deleted intermediate domain (532-1793 a.a,) and transfected it into Rev3KO-MEF. The isolated stable transformants showed comparable levels of UV-sensitivity and UV-TLS activity to those in wild-type MEF, detected using an alkaline sucrose density gradient sedimentation. These results indicate that the intermediate domain is nonessential for UV-induced translesion replication in cultured mouse cells.

Caffeine Abolishes the Ultraviolet-Induced REV3 Translesion Replication Pathway in Mouse Cells

When a replicative DNA polymerase stalls upon encountering a photoproduct on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion. Using an alkaline sucrose density gradient sedimentation technique, we previously classified this process termed UV-induced translesion replication (UV-TLS) into two types. In human cancer cells or xeroderma pigmentosum variant (XP-V) cells, UV-TLS was inhibited by caffeine or proteasome inhibitors. However, in normal human cells, the process was insensitive to these reagents. Reportedly, in yeast or mammalian cells, REV3 protein (a catalytic subunit of DNA polymerase ζ) is predominantly involved in the former type of TLS. Here, we studied UV-TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). In the wild-type MEF, UV-TLS was slow (similar to that of human cancer cells or XP-V cells), and was abolished by caffeine or MG-262. In 2 cell lines of Rev3KO-MEF (Rev3−/− p53−/−), UV-TLS was not observed. In p53KO-MEF, which is a strict control for Rev3KO-MEF, the UV-TLS response was similar to that of the wild-type. Introduction of the Rev3 expression plasmid into Rev3KO-MEF restored the UV-TLS response in selected stable transformants. In some transformants, viability to UV was the same as that in the wild-type, and the death rate was increased by caffeine. Our findings indicate that REV3 is predominantly involved in UV-TLS in mouse cells, and that the REV3 translesion pathway is suppressed by caffeine or proteasome inhibitors.

Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light‐Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation

When a replicative DNA polymerase stalls upon encountering a lesion on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion, extend by a few nucleotides, and dissociate from the 3′-OH. The replicative polymerase then resumes DNA synthesis. This process, termed translesion replication (TLS) or replicative bypass, may involve at least five different polymerases in mammals, although the participating polymerases and their roles have not been entirely characterized. Using siRNAs originally designed and an alkaline sucrose density gradient sedimentation technique, we verified the involvement of several polymerases in ultraviolet (UV) light-induced TLS in HeLa cells. First, siRNAs to Rev3 or Rev7 largely abolished UV-TLS, suggesting that these 2 gene products, which comprise Polζ, play a main role in mutagenic TLS. Second, Rev1-targeted siRNA also abrogated UV-TLS, indicating that Rev1 is also indispensable to mutagenic TLS. Third, Polη-targeted siRNA also prevented TLS to a greater extent than our expectations. Forth, although siRNA to Polι had no detectable effect, that to Polκ delayed UV-TLS. To our knowledge, this is the first study reporting apparent evidence for the participation of Polκ in UV-TLS.

Cytokinetics of a novel 1,2,3-triazene-containing heterocycle, 8-nitro-3-methyl-benzo-1,2,3,5-tetrazepin-4(3 H)-one (NIME), in the human epithelial ovarian cancer cell line OVCAR-3

The mechanism of action of the novel tetrazepinone 8-nitro-3-methyl-benzo-1,2,3,5-tetrazepin-4(3 H)-one (NIME), structurally related to the antitumour drug temozolomide, was studied in the human ovarian tumour cell line OVCAR-3. NIME preferentially inhibited DNA synthesis over protein and RNA syntheses at 3 and 24 hr post-treatment. A Maxam-Gilbert sequencing assay showed that NIME induced barely detectable levels of guanine N 7 alkylation in an isolated DNA strand, in contrast to temozolomide, a strong alkylating agent containing, like NIME, a cyclic 3-methyl-1,2,3-triazene moiety. Alkaline sucrose density-gradient sedimentation, at concentrations 2- to 10-fold lower than the ones used in the DNA sequencing assay, showed significant DNA damage in OVCAR-3 cells 24 hr after treatment with NIME. This was accompanied by a significant accumulation of cells in late S and G 2M. Cell cycle arrest was transient and was reversed after 2–3 days following drug treatment. This was in agreement with bivariate bromodeoxyuridine/propidium iodide analysis, which showed that at 100 μM, a concentration at which the majority of the cells arrested in late S and G 2M, a significant fraction of bromodeoxyuridine positive (S-phase) cells escaped the block. In an attempt to elucidate the mechanism underlying these effects, the degradation of NIME in cell culture medium was analyzed by GC–MS (gas chromatography coupled with mass spectrometry). The results showed that, in contrast to temozolomide, NIME did not convert to an open-chain alkyltriazene in cell culture medium, but to a major benzimidazole product, which exerted a minor effect on the cell cycle. This suggests that NIME, despite containing a 3-(alkyl)-1,2,3-triazene moiety, does not act by DNA alkylation but probably by generating a short-lived genotoxic species during its degradation to 6,5-benzofused derivatives.

An improved method of alkaline sucrose density gradient sedimentation to detect less than one lesion per 1 Mb DNA.

We improved alkaline sucrose density gradient sedimentation to detect very long single-strand DNA at the megabase level (from less than 1 to about 4 Mb). Hitherto, these have not sedimented correctly due to some artifacts. One artifact was aggregation of sticky DNA and proteins formed in the gradient. Then, in some gradients, biphasic distribution was observed, the major peak of which was reasonable as a result of random scission by X-rays, but the minor, fast-sedimenting population was another artifact resulting from incomplete denaturation of the DNA. We mainly reduced the centrifugal force and used a solution for cell lysis with a high concentration of salt. By means of this procedure, DNA single-strand breaks induced by relatively low doses of X-rays and subsequent repair processes can be measured in human fibroblasts. The protocol is also applicable to the study of DNA damage accompanied by strand scission, such as by UV or dimethyl sulfate as well as their repair. The technique is sensitive enough to detect even single-strand breaks induced by 0.1 J/m2 UV and sufficiently reproducible that breaks induced by increasing UV dosages were dose dependent. Thus, this technique was proven to be very sensitive, reliable and simple to perform. Therefore, this improvement will be extremely useful to investigators studying DNA repair.

Effect of ultraviolet light on DNA structure in 5-iodo-2′-deoxyuridine-substituted HSV-1 DNA

Herpes simplex virus type-1 (HSV-1) was grown in the presence of 5-iodo-2′-deoxyuridine (IdUrd), and the virion-DNA was isolated by isopycnic centrifugation in CsCl. Irradiation of IdUrd-containing HSV DNA with either 302 nm or 254 nm ultraviolet (UV) light introduced strand breakage into the DNA in a dose-dependent manner when analyzed by alkaline sucrose density gradient sedimentation. Irradiation of unsubstituted HSV DNA under similar conditions produced little strand breakage. These observations are in agreement with the proposed mechanism for photochemical generation of strand breakage in 5-halo-2′-deoxyuridine-containing DNA. Irradiation of IdUrd-substituted virions followed by analysis of the isolated DNA indicated less strand breakage than irradiation of isolated IdUrd-substituted DNA under equivalent conditions. The dosage of irradiation required to introduce DNA strand breakage in IdUrd-substituted virions was equivalent to that employed to affect >99% loss of infectious virus activity in both control and IdUrd-containing virions. It is suggested that the relative UV insensitivity of IdUrd-substituted HSV may be due to the microstructure environment of the substituted HSV DNA which may favor recombination of the photochemically formed halogenuracil radical pairs.

Mechanisms affecting peplomycin sensitivity of Chinese hamster cell lines.

Chinese hamster lung cell line V79 was ca. 13 times more resistant to peplomycin (PEP), and 6 times more resistant to bleomycin (BLM)-A2 than Chinese hamster ovary (CHO) cell line. The natural resistance of V79 cells to PEP or BLM was attributed to higher levels of BLM hydrolase activity and lower cellular uptake of the antibiotic. The sensitivity to PEP of a mutant clone CHO/O-2 T-1 was similar to that of CHO. A hybrid clone of CHO/O-2 T-1 X V79 showed an intermediate sensitivity to PEP between those of both parental cell lines, suggesting that the gene responsible for the natural resistance to PEP appears codominantly in the hybrid. The BLM hydrolase activity of the hybrid was also found intermediate between those of both parental cells. Mutant clones CHO/O-2 T-5 and CHO/O-2 T-6 were 8.3-9.0 times more sensitive to PEP than CHO cells. Hybrid clones CHO/O-2 T-5 X V79 and CHO/O-2 T-6 X V79 displayed PEP sensitivity similar to that of V79, suggesting that the gene responsible for the PEP supersensitivity (PEPss) behaves recessively in the hybrids. Both PEPss clones showed levels of BLM hydrolase and cellular uptake of [3H]PEP similar to the parental CHO cells, suggesting that the PEPss is due to neither BLM hydrolase nor cellular uptake of the antibiotic. Increased PEP-induced DNA cleavage and decreased DNA repair in the PEPss clones were demonstrated by alkaline sucrose density gradient sedimentation method. The results suggest that the PEPss of these mutant clones is attributed to decreased DNA-repairing activity and/or increased DNA-breaking activity.

Autoradiographic studies of DNA replication in Werner's syndrome cells.

We have compared cultured fibroblasts of early passage derived from patients with the Werner syndrome and from normal subjects in several aspects of cell cycle time and DNA replication. The average cycle time was prolonged in Werner's syndrome cells compared with normal cells because of changes in the duration of S phase. The durations of G1 and G2 were unchanged. In addition, the labeling index was lower in Werner's syndrome cells, suggesting that there are two cell-cycle abnormalities in Werner's syndrome cells. The cause of the prolongation of S phase was investigated by DNA fiber autoradiography and alkaline sucrose density gradient sedimentation. The rate of DNA chain elongation was not different in Werner's syndrome cells from that in normal cells, but the frequency of replication initiation was decreased in Werner's syndrome cells.

Bleomycin induced inhibition of dna synthesis in ataxia-telangiectasia cell lines

Summary Exposure of fibroblast and lymphoblastoid cell lines from normal subjects to bleomycin (30–300 μg/ml) results in a dose dependent biphasic inhibition of DNA synthesis when compared with untreated cells. Using alkaline sucrose density gradient sedimentation we have confirmed that bleomycin decreases both DNA replicon initiation and chain elongation in normal lymphoblastoid cells. Doses of up to 100 μ/ml which caused a marked decrease in the rate of DNA synthesis in normal lymphoblastoid cells had much less effect on the inhibition of DNA synthesis in lymphoblastoid cell lines from ataxia-telangiectasia patients. The inhibition of DNA replicon initiation and chain elongation in these cell lines occurred only at high doses of bleomycin (100 μg/ml).

DNA strand breaks, repair, and survival in x-irradiated mammalian cells.

The yields of unrepairable single-and double-strand breaks in the DNA of x-irradiated Chinese hamster cells were measured by low-speed neutral and alkaline sucrose density gradient sedimentation in order to investigate the relation between these lesions and reproductive death. After maximal single-strand remoining, at all doses, the number of residual single-strand breaks was twice the number of residual double-strand breaks. Both double-strand and unrepairable single-strand breaks were proportional to the square of absorbed dose, in the range 10-50 krad. No rejoining of double-strand breaks was observed. These observations suggest that, in mammalian cells, most double-strand breaks are not repairable, while all single-strand breaks are repaired except those that are sufficiently close on complementary strands to constitute double-strand breaks. Comparison with cell survival measurements at much lower doses suggests that loss of reproductive capacity corresponds to induction of approximately one double-strand break.

DNA replication in homogenates of Physarum polycephalum.

Homogenates of Physarum polycephalum incorporate [3H]dATP into nuclear DNA at an initial rate of approximately 15% of the in vivo rate. To attain this level of synthesis, cultures, are homogenized in a medium containing Mg++, EGTA, glucose and spermine. Incorporation is strongly stimulated by the addition of ATP and all four deoxyribonucleoside triphosphates to homogenates prior to incubation. Various inorganic cations other than Mg++ either do not affect synthesis or are inhibitory. Incorporation is inhibited by a nonionic detergent, Triton X-100. DNA synthesis in this cell-free nuclear system is similar in several respects to that which occurs in vivo: (1) The rate of DNA synthesis in the intact organism at a given time in the mitotic cycle is reflected by the level of synthesis in homogenates prepared from cultures at that time of the cycle; (2) DNA strands labeled in vitro exhibit alkaline sucrose density gradient sedimentation properties similar to those of daughter-strand DNA pulse-labeled in vivo; and (3) Homogenates of cultures which were pre-treated with cycloheximide incorporate [3H]dATP at about 60% of the level observed in homogenates of untreated controls.

An Improvement in Alkaline Sucrose Density Gradient Sedimentation of Mammalian Cell DNA

The usual method of alkaline (0.1-0.5M NaOH) sucrose density gradient sedimentation of intact cells enables DNA molecular weight determinations with a minimum of shearing from bacterial protoplasts. This technique however has not yet been in general successfully applied to intact mammalian cells because of higher molecular weight and contamination by protein, causing the DNA to sediment in a nonregular fashion. In addition, DNA aggregates may form which sediment to the bottom of the tube causing low recovery of the layered radioactive counts. This problem has been overcome for Ehrlich ascites cells, by including 6M guanidinium chloride in the alkaline sucrose density gradient made 0.9M in sodium hydroxide. By this method, virtually complete recovery of the radioactivity layered upon the gradient is obtained in the recovered fractions, along with a reproducible DNA peak position. Neutral sucrose gradients of Ehrlich ascites cell DNA are also possible if 6M guanidinium chloride is included in the gradient. ...