Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications

Abstract

The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell.

Figure 1.

Schematics of the used constructs for uracil detection. In our constructs, human UNG2 was used as the uracil sensor core domain. During in vitro quantification and for in situ detection, a double mutant UNG2 was created (D154N and H277N, mutated sites indicated with black lines within the schematics of the protein domains). This mutant is catalytically inactive but is still capable of binding uracil moieties in DNA (cf text for more details). The N-terminal 84 residues, responsible for the binding to RPA and PCNA, were also removed (ΔUNG) to prevent non-specific binding. The ΔUNG uracil recognizing core was fused to epitope tags (1×/3×FLAG, Au1) for immunodetection, DsRed-monomer for direct fluorescent detection and to His-tag for affinity purification.

Figure 2.

Activity and uracil binding capability of the used constructs. (A) Agarose gel electrophoresis based assay was applied to detect UNG activity. Only the hUNG2-DsRed WT construct was active on uracil-rich plasmid (indicated with an asterisk), which did not harbor the two point mutations (D154N and H277N). All other constructs used in the study do not excise uracil from DNA. (B) Uracil binding capability of the 3xFLAG-ΔUNG construct was addressed with Electrophoretic Mobility Shift Assay (EMSA). Increasing amount of the construct clearly shifts the position of the linearized vector, which is more prominent in case of uracil-rich template, indicating that the construct is capable of binding genomic uracil moieties. We have experienced similar result with the other tested constructs (Supplementary Figure S1).

Figure 3.

Design of a standard curve for in vitro quantification of genomic uracil levels. Genomic DNA isolated from log phase growing CJ236 [dut−, ung−] Escherichia coli strain was used as a standard with well-defined uracil-content during quantification. Applying a serial dilution of this standard provides a wide and reproducible range for uracil quantification. The normalized calibration curve is from four independent datasets (n = 4), where error bars show standard errors of the mean (SEM). The inset shows that the 3×FLAG-ΔUNG construct is slightly more sensitive under similar conditions, compared to the 1×FLAG-ΔUNG construct (as based on a 4 point two-third serial dilution, starting with 100 ng of standard genomic DNA).

Figure 4.

Pathways involved in thymidine synthesis in Escherichia coli, Drosophila melanogaster and humans. Key steps in dNTP synthesis focusing on the de novo thymidylate biosynthesis are shown (directly involved enzymes underlined). Dashed arrow shows pathways only present in E. coli. Inhibitors of the pathway are shown in red. 5FdUMP (5-fluorodeoxyuridylate), the metabolite of 5FU and 5FdUR, along with raltitrexed (RTX) inhibits thymidylate synthase (TYMS) while methotrexate (MTX) inhibits dihydrofolate reductase (DHFR). The enzyme responsible for dCTP-dCMP conversion in mammals is DCTPP1 (dCTP pyrophosphatase 1) (78), however, using a BLAST search, no clearcut homologue could be identified in D. melanogaster, hence we did not include it in this Figure. In E. coli, the nucleoside triphosphate pyrophosphohydrolase MAZG is responsible for this activity (79). Abbreviations are as follows: DCD: dCTP deaminase, DCTD: dCMP deaminase, DUT: dUTPase, DHF: dihydrofolate, DHFR: dihydrofolate reductase, MTHF: 5,10-methylene tetrahydrofolate, NDPK: nucleoside-diphosphate kinase, NK: nucleoside kinase, MAZG: nucleoside triphosphate pyrophosphohydrolase, NMPK: nucleoside monophosphate kinase, SHMT: serine hydroxymethyltransferase, THF: tetrahydrofolate, TYMK: dTMP kinase, TYMS: thymidylate synthase.

Figure 5.

Dot-blot assay for measuring genomic uracil levels of 5FdUR, dUR treated Escherichia coli cells. (A) CJ236 [dut−, ung−] E. coli genomic DNA was used as standard for the dot-blot assay. Quantity of genomic uracil of different drug-treated (5FdUR and dUR or both) and non-treated E. coli BL21(DE3) ung-151 samples were measured along with XL1-Blue [dut+, ung+], applied as a negative control. (B) Bar graph shows the uracil moieties/million bases of each sample (mean values ± the standard errors of the mean). Significant incrase (*) in uracil-DNA content was only observed using 5FdUR treatment, or the combined 5FdUR and dUR treatment as compared to non-treated cells (P < 0.05). Calculations were based on six independent datasets (n = 6).

Figure 6.

Dot-blot assay for measuring genomic uracil levels of Drosophila Schneider S2 cells after treatment with de novo thymidylate biosynthesis pathway inhibitors. (A) CJ236 [dut−, ung−] Escherichia coli genomic DNA was used as standard for the dot-blot assay. Genomic uracil content of different drug-treated (5FdUR, dUR or MTX, RTX, dUR) and non-treated Drosophila S2 cells were measured. (B) Bar graph shows the uracil moieties/million bases of each sample (mean values ± the standard errors of the mean). Both types of treatments led to significantly elevated genomic uracil levels as compared to non-treated cells (* = P < 0.01, ** = P < 0.05). Calculations were based on six independent datasets (n = 6).

Figure 7.

Dot-blot assay for measuring genomic uracil levels of HCT116 cells after treatment with de novo thymidylate biosynthesis pathway inhibitors and UNG inhibition. (A) CJ236 [dut−, ung−] Escherichia coli genomic DNA was used as standard for the dot-blot assay. (B) Genomic uracil levels of 5FdUR treated and non-treated HCT116 cells were measured in the contex of endogenous UNG inhibition with UGI expression. (C) Bar graph shows the uracil moieties/million bases of each sample (mean values ± the standard errors of the mean). 5FdUR treatment led to significantly elevated uracil levels only in cells also expressing UGI (* = P < 0.05) when compared to non-treated cells. Calculations were based on four independent datasets (n = 4). (D) Western blot showing GFP expression of cells transfected by the UGI-GFP vector and the empty vector used as a control (only expressing GFP). The membrane were also developed against actin as a loading control.

Figure 8.

In situ genomic uracil detection in Escherichia coli using an immunocytochemistry approach. The genomic uracil content of CJ236 [dut−, ung−] E. coli cells was visualized with the Flag-ΔUNG-DsRed construct. As a negative control, XL1-Blue cells [dut+, ung+] were also used in the same staining procedure. Only the CJ236 [dut−, ung−] E. coli sample showed staining, either detected directly through the signal of DsRed (red) or through the FLAG epitope tag (green). DAPI was used to counterstain DNA. Scale bar represents 10 μm.

Figure 9.

Detection of uracil-rich and normal plasmid DNA aggregates in MEF [ung-/-] cells. (A) Schematic image of the cytoplasmic plasmid aggregates visualized by the Flag-ΔUNG-DsRed construct. (B) Asterisks (*) show plasmid aggregates. Only cells transfected with uracil-rich plasmids could be visualized both through the DsRed (red) tag and the FLAG epitope tag (green). The DAPI staining is oversaturated to show the faint DAPI positive plasmid aggregates in the cytoplasm. Scale bar represents 10 μm.

Figure 10.

Schematics summarizing the developed in vitro quantification and in situ detection method. (A) Schematic image of the applied in vitro quantification through a dot-blot approach. (B) Schematic image of the applied immunocytochemical approach for in situ uracil detection.