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  • crth2 antagonist In order to assess quantitatively the contr

    2019-10-16

    In order to assess quantitatively the contribution of the ung, dug and dut crth2 antagonist to uracil-DNA accumulation in E. coli, we have developed a novel method for measuring uracil residues in DNA that is based on the approach of Kow and co-workers [33] for measuring AP-sites in DNA. The method, which we call the Ung-ARP assay, utilizes the Aldehyde reactive probe (ARP) reagent [33], an alkoxy amine attached via a flexible linker to a biotin moiety, to tag freshly created 3′-α,β-unsaturated aldehydes in DNA with biotin. The DNA aldehydes are formed by T4 pyrimidine dimer DNA glycosylase/AP-lyase cleavage of AP-sites [34] resulting from Ung-mediated uracil excision. Using this method, we analyzed the uracil content of DNA isolated from four E. coli strains, isogenic except with respect to ung and dug, and examined the effect of growth phase, nucleoside supplementation, and 5-fluoro-2′-deoxyuridine, an inhibitor of thymidylate synthase, on uracil accumulation. In addition, levels of uracil were determined in the DNA of E. coli CJ236 (dut-1 ung-1), which is defective in uracil-DNA glycosylase activity and has reduced, temperature sensitive, dUTP pyrophosphatase activity.
    Materials and methods
    Results
    Discussion In this report, the influence of the ung, dug, and dut genes in preventing uracil residues from accumulating in E. coli DNA was quantitatively determined using a novel assay, the Ung-ARP assay. We found that inactivation of the ung gene in CY10 resulted in a more than 30-fold increase in the level of uracil-DNA, whereas inactivation of dug had no measurable effect. Uracil levels in the DNA of CY11, an ung dug mutant, were not significantly higher compared to the ung mutant. Inactivation of ung and defective dut in the E. coli strain CJ236 resulted in a 100-fold increase in levels of uracil-DNA compared to the inactivation of ung alone in CY10. We observed that a dug mutation in an ung+ background had no detectable effect on uracil levels relative to the wild type strain (Fig. 3). This result was consistent with the report by Mokkapati et al. [17] which concluded that Ung and Dug process different lesions in vivo. Since extracts of ung dug strains lack detectable uracil-initiated BER [16], it would appear that Ung and Dug are the sole activities in E. coli capable of removing uracil from DNA. Thus, use of CY11 allows determination of the amount of uracil that escaped cleavage by dUTP pyrophosphatase and was incorporated into nascent DNA under various growth conditions [3]. We consistently observed that the level of uracil in DNA extracted from saturated cultures of CY11 was lower than that from early log cultures (Fig. 3B). This reduction may result from slowing of cell division as the cell density of the culture reaches a critical level and nutrients become limiting. Longer periods between replication cycles may give dUTP pyrophosphatase more time to sanitize DNA precursor pools. In addition, as E. coli cultures approach saturation, some cells die and release their contents, which include nucleosides and nucleotides, into the culture medium. These compounds can be utilized by salvage pathways in living cells to supplement dTMP pools. Levels of uracil in CY11 cells treated with 5-fluoro-2′-deoxyuridine declined in saturated phase (Fig. 4). One possible explanation for this decline is that the amount of 5-fluoro-2′-deoxyuridine in the medium was nearly depleted in saturated phase cultures. Summers and Raksin [61] found that by supplementing the culture medium with adenine, guanosine, cytidine, and uridine, single-step resistance to 5-fluoro-2′-deoxyuridine (50μM) could be obtained by inactivation of the gene for thymidine kinase, tdk. While we did not look for tdk or deoA mutations in CY11 grown in the presence of 5-fluoro-2′-deoxyuridine, we note that the amount of 5-fluoro-2′-deoxyuridine (50μM=12.3μg/ml) used by Summers and Raksin to induce tdk mutation was 615-fold greater than the highest concentration used in this investigation.