Production of clastogenic DNA precursors by the nucleotide metabolism in Escherichia coli

Abstract

RdgB is a bacterial dNTPase with a strong in vitro preference for non-canonical DNA precursors dHapTP, dXTP and dITP that contain deaminated or aminogroup-modified purines. Utilization of these nucleotides by replisomes in rdgB mutants of Escherichia coli produces modified DNA, on which EndoV nicking near the base analogues initiates excision repair. Some EndoV-initiated excision events cause chromosomal fragmentation, which becomes inhibitory if recombinational repair is also inactivated (the rdgB recA co-inhibition). To reveal the sources and the identities of the non-canonical DNA precursors, intercepted by RdgB in E. coli, we characterized 17 suppressors of the rdgB recA co-inhibition. Ten suppressors affect genes of the RNA/DNA precursor metabolism, identifying the source of non-canonical DNA precursors. Comparing chromosomal fragmentation with the density of EndoV-recognized DNA modifications distinguishes three mechanisms of suppression: (i) reduction of the non-canonical dNTP production, (ii) inhibition of the base analogue excision from DNA and (iii) enhancement of the cell tolerance to chromosomal fragmentation. The suppressor analysis suggests IMP as the key intermediate in the synthesis of the clastogenic DNA precursor, most likely dITP.

Fig. 1. Deaminated or amino group-modified base analog toxicity and the expected types of suppressors of the rdgB recA co-inhibition

Hap, 6-N-hydroxylaminopuriune; ba, base analog; nc-, non-canonical (for dNTP or dNMP). Stages are identified by the genes. Numbers in the stars denote the segment of the metabolic scheme where suppressors are possible: #1, decreased production of nc-dNTP; #2, increased alternative interception of nc-dNTP; #3, decreased excision of the base analog from DNA; #4, slowing down replication or accelerating the excision repair; #5, ameliorating the recombinational repair defect of the recA(Ts) mutant; #6, tolerance to double-strand DNA breaks.

Fig. 2. Isolation of suppressors of the rdgB recA co-inhibition and quantification of their effect on growth of the double mutant

A. Spotting serial dilutions of cultures grown at 28°C and subsequently incubating them at 43°C reveals the rdgB recA(Ts) synthetic inhibition, as well as its suppression. The strains are: WT, AB1157; rdgB recA, (JB30); rdgB recA deoB, (BB004); rdgB recA dusB, (BB020); rdgB recA hpt, (BB028); rdgB recA dgt, (BB036). B. Steps of the protocol used for quantification of growth inhibition in the double mutant and the extent of its suppression: 1) a saturated overnight culture grown at 28°C is diluted 10⁻⁵-fold; 2) 10 μl spots are incubated on an LB plate at 28°C or 42°C for 17 hours, 3) agar stubs with the spotted cells are cut out of the plate and resuspended in saline for 1 hour; 4) the cell suspensions are serially diluted, spotted by 10 μl onto LB agar and incubated at room temperature to determine the titer for each spot at the original temperatures. The titer of growth at 42°C divided by the titer of growth at 30°C (28°C) represents “growth” or its inhibition and is reported in “C”. C. Quantification of growth inhibition and its suppression by the protocol illustrated in “B”. The values are means of 4 to 20 independent measurements ± SE. D. Additional copies of the functional genes, identified by the most numerous suppressors, further decrease viability of the double rdgB recA mutant. pK80, low copy-number vector; pBB13, pK80 carrying nfi, deoB, deoD, hpt, purR, dgt, and tdk functional genes.

Fig. 3. The “missing” suppressors and the effect of slow growth

A. A scheme of xanthine (X) and hypoxanthine (H) conversion into nucleotides. R-1-P, ribose-1-phosphate; R-5-P, ribose-5-phosphate; PRPP, 5-phosphorybosyl-1-pyrophosphate. Isolated inactivational and overproductional suppressors on the “hypoxanthine side” are indicated by spiked and smooth ovals, correspondingly. The corresponding genes on the “xanthine side” (guaA, gpt and xapA) are in bold. B. The effect of “missing suppressors” on the degree of rdgB recA co-inhibition. The values are means of four-five independent measurements ± SE. C. Slowing the overall metabolism with chloramphenicol marginally increases growth of the double mutant. The values are means of four independent measurements ± SE (in most cases, masked by the size of the symbols). D. Slowing growth of wild type cells with thinner media marginally increases growth of the double mutant. 20 AA, all aminoacids added, according to the recipe of Neidhardt (Neidhardt et al., 1974); 6 AA, the six aminoacids that AB1157 requires (arginine, glutamine, histidine, leucine, proline, and threonine) are added. The values are means of three independent measurements ± SE.

Fig. 4. Testing the suppression of the recA defect

A. The effect of suppressors on UV-sensitivity of a single recA200 mutant at 42°C. Serial dilutions were spotted on LB agar plates and irradiated with a single dose of 12 J/m². The data are means of four independent measurements ± SE. B. Suppression does not ameliorate the recA(Ts) defect. All the suppressors were represented by their growth at 42°C versus 28°C in the rdgB recA(Ts) double mutant (X axis), as well as by their survival of UV light in the recA(Ts) single mutant (Y axis). Wild type for both axes is AB1157; (−) control is rdgB recA(Ts) (JB30) for the X-axis, recA(Ts) (JC9941) for the Y-axis. Error bars are from the corresponding assays.

Fig. 5. Epistatic analysis of the weak suppressors

Suppressor mutations were combined pairwise in the rdgB recA(Ts) background, and the effect of their combination on the viability of the strain at 42°C was compared to the “expected” multiplicative effect of the double mutation (product of the effects of the corresponding single mutations). In each group of bars, the effects of single mutations and the calculated effect of the double mutation are normalized to the viability of the original rdgB recA(Ts) mutant and then compared with the observed effect of the double mutation. The double mutant values are averages of four independent measurements. Error bars are not shown because the averages are normalized. A. Three out of four possible combinations with purR show strong synergy — the observed effect is more than 10-fold higher that the multiplicative one. B. The pairwise combinations between putA, rpoC and dgt tend to be either weakly synergistic (less than 10-fold higher than the multiplicative effect) or additive (= multiplicative: the effect of the double mutant roughly equals the product of the effects of the single mutants). C. All four combinations with tdk show either additivity or weak epistasis.

Fig. 6. The effect of suppressors on chromosomal fragmentation in the rdgB recBC double mutant

A. A representative PFGE showing chromosomal fragmentation for each control strain and four suppressors. CZ, compression zone. Intact chromosomes remain in the wells at the origin of the gel, while linear sub-chromosomal fragments migrate into the lanes. B. Quantification of chromosomal fragmentation. Values are means of 6–17 independent measurements ± SE. The wild type (AB1157) and dut recBC(Ts) (AK107) strains are (−) and (+) controls for fragmentation (Kouzminova et al., 2004). C. Plotting growth (X) versus fragmentation (Y) reveals two groups of suppressors. All the suppressors were represented by their growth at 42°C versus 28°C in the rdgB recA(Ts) double mutant (X axis), as well as by their fragmentation in the rdgB recBC(Ts) double mutant (Y axis). Wild type is AB1157 for the X-axis, recBC(Ts) (SK129) for the Y-axis; (−) control is rdgB recA(Ts) (JB30) for the X-axis, rdgB recBC(Ts) (JB36) for the Y-axis. Error bars are from the corresponding assays.

Fig. 7. The effect of suppressors on the density of EndoV-recognized DNA modifications

A. Assay for EndoV-sensitivity of a supercoiled plasmid (the conversion between supercoiled and relaxed circular forms in a 22.5 kbp plasmid). B. Quantification of EndoV-sensitivity. The density of EndoV-recognized DNA modifications in each suppressor in the rdgB background is shown at the percentage of a 22.5 kbp plasmid that is nicked after being treated with EndoV. Values are means of 4–16 independent measurements ± SE. The wild type (AB1157) is the background, the rdgB single mutant (JB29) shows the elevated levels of EndoV-recognized DNA modifications, while the rdgB nfi double mutant (BB112) shows further elevation due to the additional excision repair defect. C. Plotting growth (X) versus EndoV-sensitivity (Y) reveals two groups of suppressors. All the suppressors were represented by their growth at 42°C versus 28°C in the rdgB recA(Ts) double mutant (X axis), as well as by EndoV-sensitivity of their DNA in the rdgB single mutant (Y axis). Wild type is AB1157 for both axes; (−) control is rdgB recA(Ts) (JB30) for the X-axis, rdgB (JB29) for the Y-axis. Error bars are from the corresponding assays.

Fig. 8. Summary of suppressors and the identity of the RdgB-intercepted non-canonical dNTP, revealed by a tentative pathways of its biosynthesis

A. Summary: plotting fragmentation (X) versus EndoV-sensitivity (Y) reveals three groups of suppressors. This is the third possible pairwise comparison, of the types presented in Fig. 4C and 5C. All the suppressors are represented by their fragmentation in the rdgB recBC(Ts) double mutant (X axis), as well as by EndoV-sensitivity of their DNA in the rdgB single mutant (Y axis). Wild type is recBC(Ts) (SK129) for the X-axis, AB1157 for the Y-axis; (−) control is rdgB recBC(Ts) (JB36) for the X-axis, rdgB (JB29) for the Y-axis. Error bars are from the corresponding assays. B. A scheme of the nucleotide metabolism to explain our suppressor results. R-1-P, ribose-1-phosphate; R-5-P, ribose-5-phosphate; PRPP, 5-phosphorybosyl-1-pyrophosphate; Hx, hypoxanthine. Inactivational and overproductional suppressors are indicated by spiked and smooth ovals, correspondingly. The only hypothetical step (Gmk-catalyzed IMP—>IDP conversion) is shown by a broken arrow.