aphidicolin and Bloom-Syndrome

Perturbed DNA replication either activates a cell cycle checkpoint, which halts DNA replication, or decreases the rate of DNA synthesis without activating a checkpoint. Here we report that at low doses, replication inhibitors did not activate a cell cycle checkpoint, but they did activate a process that required functional Bloom's syndrome-associated (BLM) helicase, Mus81 nuclease and ataxia telangiectasia mutated and Rad3-related (ATR) kinase to induce transient double-stranded DNA breaks. The induction of transient DNA breaks was accompanied by dissociation of proliferating cell nuclear antigen (PCNA) and DNA polymerase alpha from replication forks. In cells with functional BLM, Mus81 and ATR, the transient breaks were promptly repaired and DNA continued to replicate at a slow pace in the presence of replication inhibitors. In cells that lacked BLM, Mus81, or ATR, transient breaks did not form, DNA replication did not resume, and exposure to low doses of replication inhibitors was toxic. These observations suggest that BLM helicase, ATR kinase, and Mus81 nuclease are required to convert perturbed replication forks to DNA breaks when cells encounter conditions that decelerate DNA replication, thereby leading to the rapid repair of those breaks and resumption of DNA replication without incurring DNA damage and without activating a cell cycle checkpoint.

Topics: Aphidicolin; Bloom Syndrome; Cells, Cultured; Comet Assay; DNA; DNA Breaks, Double-Stranded; DNA Helicases; DNA Replication; DNA-Binding Proteins; Dose-Response Relationship, Drug; Endonucleases; Fibroblasts; Fluorescent Antibody Technique, Direct; Histones; Humans; Hydrogen Peroxide; Kinetics; Phosphorylation; Proliferating Cell Nuclear Antigen

Bloom's syndrome (BS) is an autosomal recessive disorder associated with an elevated incidence of cancers. The gene mutated in BS, BLM, encodes a RecQ helicase family member. BS cells exhibit genomic instability, including excessive homologous recombination and chromosomal aberrations. We reported previously that BS cells also demonstrate increased error-prone nonhomologous endjoining, which could contribute to genomic instability in these cells. Here, we show that BS cells display an abnormality in the timing of replication of both early-replicating genes and late-replicating loci such as chromosomal fragile sites. This delayed replication is associated with a constitutively increased frequency of sites of DNA damage and repair, as determined by the presence of DNA repair factors such as RAD51 and Ku86. In addition, another RecQ family helicase, WRN, also localizes to these repair sites. The presence of these repair sites correlates with the temporal appearance of cyclin B1 expression, indicative of the cells having progressed beyond mid-S phase in the cell division cycle. Critically, these defects in BS cells are the direct result of loss of BLM function, because BS cells phenotypically 'reverted' following transfection with the BLM cDNA no longer show such defects. Thus, our data indicate that constitutive DNA damage is coupled to delayed DNA replication in BS cells.

Topics: Aphidicolin; Bloom Syndrome; DNA Damage; DNA Repair; DNA Replication; Humans; S Phase

In Bloom's syndrome (BS) the regulation of uracil-DNA glycosylase, an enzyme involved in the repair of DNA containing 5-FU, is altered. 5-FU induces higher levels of DNA fragmentation in BS cells than in non-BS cells. The increase in DNA fragmentation is connected to the cytotoxic mechanism where 5-FU is incorporated into DNA. When 5-FU induces DNA fragmentation by a mechanism not involving the incorporation of drug into DNA, the levels of DNA fragmentation in BS and non-BS cells remain similar.

Topics: Aphidicolin; Bloom Syndrome; Cell Cycle; Cell Line; Diterpenes; DNA; DNA Damage; DNA Glycosylases; DNA Repair; Fluorouracil; Humans; N-Glycosyl Hydrolases; Sulfonamides; Uracil-DNA Glycosidase

Bloom's syndrome (BS) cells display a characteristic genomic instability, notably an elevated frequency of sister-chromatid exchange. Replicating DNA in cultured BS cells was labeled with [3H]thymidine using several time schedules. Separation of DNA in agarose gels showed high molecular weight DNA and three classes of DNA replication intermediates: 20-kilobase DNA, 10-kilobase DNA, and Okazaki fragments. In contrast newly replicated DNA from normal cells showed no 20-kilobase DNA replication intermediates. Certain BS cells, exceptional in that their characteristic genomic instability has for unknown reasons been corrected, also differed from normal cells in having the 20-kilobase intermediate, but they differed from both normal cells and the other (the uncorrected) BS cells in lacking the 10-kilobase DNA replication intermediates.

Topics: Aphidicolin; Bloom Syndrome; Cell Line; Diterpenes; DNA Replication; Humans; Molecular Weight; Sister Chromatid Exchange; Time Factors

Repair-proficient human cells can be sensitized to exposure to UV radiation at 254 nm by postirradiation incubation in the presence of the eukaryotic alpha polymerase inhibitor, aphidicolin. The degree of sensitization has been examined in cells cultured from humans suffering from various types of sun-sensitive syndromes. Xeroderma pigmentosum (XP) variant and Bloom's cell lines (both excision proficient) were strongly sensitized by aphidicolin. An excision repair proficient Cockayne's cell line and a deficient XPD line were both sensitized to a level similar to the sensitivity of excision deficient XPA cells. In contrast, three XPC cell lines which show intermediate UV-induced repair replication and UV sensitivity were sensitized little (in one case) or not at all (in two cases) to UV by postirradiation inhibition of the alpha polymerase. These results lead us to conclude that there are two independent pathways of biologically effective excision repair, the major one of which involves the alpha polymerase and a second, less efficient and slower pathway which is independent of the alpha polymerase and which is the only pathway operating in two of the three XPC strains tested. The rates of biologically effective excision repair were similar in normal, XP variant, and Cockayne's cell lines, but these rates were considerably higher than published rates of dimer excision measured under similar conditions.

Topics: Aphidicolin; Bloom Syndrome; Cells, Cultured; Diterpenes; DNA Polymerase II; DNA Repair; Humans; Sunlight; Ultraviolet Rays; Xeroderma Pigmentosum

Ultraviolet radiation induced more unscheduled DNA synthesis (UDS) in ten Bloom syndrome (BS) fibroblast strains than in control cells, but this difference could be suppressed by aphidicolin treatment in at least nine BS strains. Aphidicolin, 1 and 5 micrograms/ml, were required to inhibit by 30% the UDS of BS and control cells respectively, but the DNA replication of BS cells did not prove abnormally sensitive to such an inhibitor. These findings are discussed in relation to current knowledge of the action of aphidicolin and hypotheses of the metabolic defect in BS.

Topics: Aphidicolin; Bloom Syndrome; Cells, Cultured; Diterpenes; DNA; DNA Polymerase II; DNA Repair; DNA Replication; Fibroblasts; Humans; In Vitro Techniques; Nucleic Acid Synthesis Inhibitors; Ultraviolet Rays