We also determined the uracil content of

We also determined the uracil content of CJ236 DNA by thin-layer chromatography of in vivo32P- or [6-3H]uracil-labeled CJ236 2′-deoxyribose 5′-monophosphates as well as by LC/MS of deoxyuridine and thymidine 2′-deoxynucleosides (Table 1, Table 2, Table 3). Examination of the 32P data showed that, based on the total 32P radioactivity detected, the amount of 32P dUMP observed corresponded to 6644±1179U/106nt. However, the ratio of 32P dUMP to the sum of 32P dUMP+32PdTMP, 0.0372, indicated that level of uracil was 9300±725U/106nt. The reason for this disparity is not clear; however, it is apparent the distribution of 32P radioactivity in spots corresponding to dAMP, dCMP, dGMP, and dTMP was skewed toward dGMP, which represented almost 36% of total 32P cpm. Analysis of [6-3H]uracil-labeled CJ236 showed that the ratio of 3H dUMP to the sum of 3H dUMP+3H dTMP was 0.0754,which, upon extrapolation, corresponds to 18,850U/106nt (Table 2). This determination of uracil levels in CJ236 DNA was about two-fold higher than that obtained with 32Pi labeling, most likely because the dUTP pool was increased by [6-3H]uracil supplementation through the action of the A-443654 salvage pathway [66]. Using LC/MS we found that the U to T ratio was 0.032, which was equivalent to 7752U/106nt (Table 3). Given that labeling with [6-3H]uracil may have produced artifactually high U:T ratios, the average level of uracil residues in CJ236 DNA detected by 32Pi labeling and LC/MS was 7899U/106nt, compared to 3000U/106nt by the Ung-ARP assay, a ∼2.5-fold difference. We speculate that the extremely high concentration of uracil residues in dut-defective bacteria like CJ236 DNA may have been underestimated by the Ung-ARP assay because (1) not all uracil residues may have been accessible for excision by uracil-DNA glycosylase, and (2) not all the biotin-tagged sites in the DNA deposited on the nitrocellulose membrane were accessible to streptavidin binding. Recent measurements of uracil levels in the DNA of folate-deficient human beings range from 4.6μmolU/mol DNA (4.6U/106nt) [29] to 34,600–167,000 uracils/diploid cell [67] to 500,000–4,000,000 uracils/diploid cell (83.3–667U/106nt) [32]. Since the reproducibility of the Ung-ARP assay for uracil levels in the low to medium range was excellent, we propose that this method is well suited for determining the level of uracil residues in human cells.
Acknowledgments
Introduction Deoxyuridine 5′-triphosphate pyrophosphatase (dUTPase, EC3.6.1.23) plays an essential role in the nucleotide metabolism of all organisms by catalyzing the hydrolysis of dUTP to dUMP and pyrophosphate [1], [2]. dUTPase serves two major biological functions. By hydrolyzing dUTP, it helps to maintain a low dUTP:dTTP ratio, thereby reducing the rate of dUTP incorporation into DNA. The second role of dUTPase is to generate the precursor dUMP for the biosynthesis of thymidine nucleotides [3]. Amino acid sequence analyses display five conserved motifs within relatively short subunits of about 150 residues (although exceptions occur among viruses and parasites). Four very similar crystal structures of dUTPases, from eubacteria, retroviruses and man, show a unique homotrimeric arrangement, where each of the three active sites are formed by contributions from all three subunits; motifs 1, 2 and 4 from subunit A, motif 3 from subunit B and motif 5 from subunit C [2], [4], [5], [6]. Most DNA polymerases cannot distinguish between thymine and uracil. However, family B DNA polymerases from Archaea, such as Pfu DNA polymerase, strongly bind to template strand uracil and stall polymerization in response to this base [7], [8], [9]. The main inhibitor of PCR with Pfu DNA polymerase is dUTP generated by the deamination of dCTP at high temperatures [10]. Pyrococcus furiosus dUTPase is added to commercial preparations of thermostable DNA polymerases as an enhancing factor (Pfu Turbo DNA polymerase). By scavenging dUTP from the PCR mixture, the thermostable enzyme enables synthesis of long DNA fragments (>10kb) [10].