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  • Addition of deoxyuridine to CY cultures led to a


    Addition of deoxyuridine to CY11 cultures led to a significant decline in the amount of uracil-DNA (Fig. 5). Deoxyuridine effectively induces the deo operon and is efficiently degraded to uracil and deoxyribose 1-phosphate by the deoA gene product, thymidine phosphorylase; a minor percentage of deoxyuridine is phosphorylated to dUMP by thymidine kinase and available for methylation by thymidylate synthase [62]. Since deoxyuridine acts a competitive inhibitor of thymine phosphorylase-mediated cleavage of thymidine, more thymidine is available for phosphorylation to dTMP. In addition, expansion of the deoxyribose 1-phosphate pool enables production of thymidine from endogenous thymine in a synthetic reaction catalyzed by thymidine phosphorylase [62]. The dUTP pool is probably reduced by deoxyuridine supplementation, since the increase in the dTTP pool inhibits dCTP deaminase, an important contributor to the dUTP pool [10]. Uridine can not serve as a source of deoxyribose 1-phosphate, and thymidine is rapidly broken down to thymine and deoxyribose 1-phosphate, which in turn is degraded by phosphodeoxyribo-mutase (deoB) and phosphodeoxyribo-aldolase (deoC). As observed by Budman and Pardee [52] and here in Fig. 5, incorporation of exogenous thymidine into DNA is increased by uridine, presumably because uridine competitively inhibits thymidine phosphorylase and prevents its induction by thymidine. DNA sequencing of the CJ236 gene revealed that the defective dUTP pyrophosphatase activity in this strain was the result of a Thr24Ile amino ω-Agatoxin TK kinase change. In the crystal structure of E. coli dUTP pyrophosphatase solved by Barabas et al. [63], the oxygen atom of the Thr24 side chain (Thr25 as shown in Fig. 8) shares a hydrogen bond with a water molecule that is hydrogen-bonded to a carboxylate oxygen of the Asp90 side chain. Since Asp90 is responsible for coordinating the water molecule (Wcat in Fig. 8) that initiates the nucleophilic attack on the α-phosphorus atom of dUTP, it important that the Asp90 side chain be maintained in the correct position, and the Thr25 oxygen contributes indirectly to this task. Substitution of Thr24 with Ile introduces an apolar residue at this position, which apparently is poorly tolerated in terms of enzyme activity. While Thr24 is not strictly conserved in dUTP pyrophosphatases, an apolar residue has not been found at this sequence position [64]. This observation argues for the importance of polar/charged character at this location in the enzyme/substrate structure. In eukaryotic dUTP pyrophosphatase, the position equivalent to Thr24 is occupied by Ser, whereas in viral enzymes, either Thr or Ser is found. Mutation of Thr24 in E. coli dUTP pyrophosphatase to a polar residue such a His (as occurs in Mycobacteria), which is also capable of hydrogen bonding, may result in retention of enzyme activity by some rearrangement around the Asp90 side chain. AP-sites inhibit uracil-DNA glycosylase because the enzyme binds more tightly to the AP-site reaction product than to uracil-DNA [59], [65]. Thus, when our initial determination of the amount of uracil in CJ236 (dut-1ung-1) DNA by the Ung-ARP assay appeared low (1000U/106nt), we suspected that Ung was AP-site inhibited and increased the Ung:uracil-DNA molar ratio by reducing the amount of substrate DNA and holding the amount of enzyme constant. Reducing the amount of CJ236 DNA in the assay 10- and 100-fold produced results that were in close agreement: 2915 and 3005U/106nt, respectively. AP-site inhibition of Ung by other DNAs, such as CY11 (ung dug), was not observed (data not shown). Blount and Ames [25] reported that E. coli CJ236, propagated at 37°C, contained 1.22% uracil residues by weight, as determined by HPLC reverse chromatography and UV absorbance. We calculate that 1.22% uracil by weight is equivalent to 12.2μg uracil per mg of DNA, 14% replacement of T by U, or ∼35,000U/106nt. In a subsequent report, Atamna et al. [30] determined by GC/MS that the amount of uracil in CJ236 DNA was actually 0.28%, which we calculate to be ∼8000U/106nt. The effect of temperature on dUMP incorporation in an E. coli dut ung strain was investigated by Warner and co-workers, who propagated BD1157 (dut-1 ung-1) at different temperatures in the presence of [6-3H]uridine [8]. By interpolation of their graphical data, we estimate that replacement of thymine with uracil in BD1157 DNA increased from 5% at 30°C to over 12% at 37°C [8]. Thus, the amount of uracil in E. coli dut-1 DNA can vary considerably depending on the growth temperature.