Sources of thymidine and analogs fueling futile damage-repair cycles and ss-gap accumulation during thymine starvation in Escherichia coli

Abstract

Thymine deprivation in thyA mutant E. coli causes thymineless death (TLD) and is the mode of action of popular antibacterial and anticancer drugs, yet the mechanisms of TLD are still unclear. TLD comprises three defined phases: resistance, rapid exponential death (RED) and survival, with the nature of the resistance phase and of the transition to the RED phase holding key to TLD pathology. We propose that a limited source of endogenous thymine maintains replication forks through the resistance phase. When this source ends, forks undergo futile break-repair cycle during the RED phase, eventually rendering the chromosome non-functional. Two obvious sources of the endogenous thymine are degradation of broken chromosomal DNA and recruitment of thymine from stable RNA. However, mutants that cannot degrade broken chromosomal DNA or lack ribo-thymine, instead of shortening the resistance phase, deepen the RED phase, meaning that only a small fraction of T-starved cells tap into these sources. Interestingly, the substantial chromosomal DNA accumulation during the resistance phase is negated during the RED phase, suggesting futile cycle of incorporation and excision of wrong nucleotides. We tested incorporation of dU or rU, finding some evidence for both, but DNA-dU incorporation accelerates TLD only when intracellular [dUTP] is increased by the dut mutation. In the dut ung mutant, with increased DNA-dU incorporation and no DNA-dU excision, replication is in fact rescued even without dT, but TLD still occurs, suggesting different mechanisms. Finally, we found that continuous DNA synthesis during thymine starvation makes chromosomal DNA increasingly single-stranded, and even the dut ung defect does not completely block this ss-gap accumulation. We propose that instability of single-strand gaps underlies the pathology of thymine starvation.

Keywords: Base excision repair; Chromosomal fragmentation; Chromosomal replication; DNA-dU incorporation; Ribonucleotide excision repair; Thymineless death.

Fig. 1.. The metabolism of dTTP production, the phenomenon of TLD, the futile cycles, and the expected changes in nucleotide metabolism mutants and kinetics of chromosomal fragmentation.

A. The metabolic pathways. Compounds of DNA metabolism are in black, compounds of RNA metabolism are in orange. Genes are colored according to functions: general (blue), dTTP-synthesis (purple) or DNA repair (magenta). The big arrow shades are: green, general biosynthesis; yellow, salvage; blue, DNA degradation; cyan, RNA degradation; magenta, futile DNA-dU misincorporation/excision cycle; orange, futile DNA-rU misincorporation/excision cycle. B. Extended time course of the culture titer during thymine starvation in our standard conditions, to highlight the survival phase. The strains are: ThyA+, KKW59; thyA, KKW58. The three phases of TLD are shown in color: green for the resistance phase, red for the RED phase, purple for the survival phase. The values in this and subsequent figures are means of 4–40 independent measurements ± SEM. C. DAPI-staining of cells grown in the presence of dT, as well as the same cells T-starved for 1 hour or for 3 hours. D. The thyA recBCD mutants lack the resistance phase, while the thyA recF mutants die slowly. The strains are: thyA, KKW58; thyA recBCD, KJK63; thyA recF, RA31. Here and henceforth: if error bars are not visible, they are masked by the symbols. E. A scheme of the futile fork-break-and-repair cycle. F. Various models of replisomes traversing A-runs in the template DNA during T-starvation that we tested in this work.

Fig. 2.. Testing the intrachromosomal DNA redistribution idea.

A. A pulsed-field gel showing kinetics of chromosome fragmentation induced by thymine starvation. LMW, low molecular weight species, ~50–200 kbp in size. The strain is KKW58. B. Quantitative kinetics of chromosomal fragmentation, from several gels like in “A”. The data are means (n = 4 or 5) ± SEM. C. The scheme of how cannibalizing linear DNA (magenta arcs) generated by disintegration of some of the stalled forks (orange circles) could allow cells to keep the remaining forks active (green circles), explaining the resistance phase. D. Cell counts in T-starved cultures of the thyA mutant (KKW58) normalized to time of dT removal. E. Stabilization of prelabeled chromosomal DNA (TCA-precipitable counts) in UV-irradiated recA mutant cells by recBC sbcBC defect. The strains are: recA, RA46; recA recBCD sbcBC, RA47. The UV-dose was 50 J/m². F. Time course of TLD in the recBCD sbcBC thyA mutant. The strains are: thyA, KKW58; recBCD sbcBC thyA, RA45. G. Change in the total amount of chromosomal DNA over time (“chromosomal DNA evolution”) in the thyA mutant (KKW58) during T-starvation. The first two phases of T-starvation are shown in the same colors as in Fig. 1A. H. Expectations about the origin and terminus copy number evolution during T-starvation according to the intrachromosomal DNA redistribution idea. I. Evolution of the amount of origin and terminus under the same conditions as in “G”.

Fig. 3.. Testing the contribution of ribo-thymine from stable RNA.

A. A scheme of stable RNAs (tRNA has one rT, 23S rRNA has two) and how many thymine residues they can yield per average rapidly-growing cell. B. Time course of TLD after 15’ @ 54°C heat shock. The strain is KKW58. In this case, the experiment-specific thyA TLD curve was used. C. Stability of tRNA and rRNA during thymine starvation. The strain is KKW58. Stability is expressed as the ratio of the corresponding RNA species in the cells incubated without dT to cells from the same culture incubated in the presence of dT. The actual measurements, normalized to time 0, are shown in Figures S3B and S3C. D. Recruitment of rT by degrading stable RNA kills two birds with one stone: it yields dT to support stalling replication forks and at the same time it inhibits translation, blocking new initiations and slowing down general metabolism. Both changes contribute to metabolism rebalancing. E. Time course of TLD in the thyA trmA mutant (RA9). F. Time course of TLD in the thyA rumA rumB mutant (RA13). G. Time course of TLD in the trmA thyA and trmA rumB thyA mutants. The strains are: thyA, KKW58; trmA thyA, RA9 (from “E”); trmA rumB thyA, RA14. H. Evolution of the chromosomal DNA absolute amount in the thyA and trmA thyA mutants (strains like in “E”) during T-starvation.

Fig. 4.. The role of deoxy-uridine in TLD.

A. A scheme of DNA-dU misincorporation and its consequence in thyA, ung thyA, dut thyA and dut ung thyA mutants undergoing thymine starvation. Green fill, growth with dT. Light pink fill, attempted replication without dT using little dU (Dut+) with active DNA-uracil removal (Ung+). Yellow fill, replication without dT using little dU (Dut+), with no DNA-uracil removal (ung). Pink fill, attempted replication without dT but with plenty of dU (dut) with active DNA-uracil removal (Ung+). Light green fill, normal replication without dT but with plenty of dU (dut) and without DNA-uracil excision (ung). B. Time course of TLD in ABS-endo mutants. Strains are: thyA, KKW58; xthA thyA, RA22; nfo thyA, RA23; xthA nfo thyA, RA24. C. Time course of TLD in the dut thyA and ung thyA mutants. The +dT/-dT medium switch in this case was by centrifugation. Strains are: thyA, KKW58; dut thyA, RA16; ung thyA, KJK78; dut ung thyA, RA18. D. Evolution of the chromosomal DNA amount in the thyA (KKW58) and dut ung thyA (RA18) mutants during thymine starvation. E. Evolution of the replication origin copy number in the thyA and dut ung thyA mutants (strains like in “D”) during thymine starvation. F. Evolution of the chromosomal terminus copy number in the thyA and dut ung thyA mutants (strains like in “D”) during thymine starvation. G. TLD kinetics of the recF dut ung thyA mutant. The strains are thyA,KKW58; thyA recF, RA31; thyA dut ung, RA18; thyA dut ung recF, RA32.

Fig. 5.. Density of DNA-dU in the ung thyA and dut ung thyA mutants, either starved or not for dTTP.

+dT, growth in the presence of thymidine; –dT, starvation without thymidine. Strains are: thyA, KKW58; ung thyA, KJK78; dut ung thyA, RA18. A. A representative gel (1.1% agarose) of DNA-dU density determination in plasmid DNA (pMTL20). UDG, treatment with uracil-DNA glycosylase; Exo III, treatment with exonuclease III; U + E, treatment with both UDG and Exo III. B. Quantification of the DNA-dU density (presented as frequency = 1/density) in the ung thyA mutant grown in the presence of absence of dT, from several gels like in “A”. The “–dT” culture is processed 5 hours after dT removal by filtration. There are two different conditions of growth in the presence of dT, though: “+dT #1” is also processed 5 hours after the filtration (but dT was re-added in this case), so the culture becomes stationary. In contrast, “+dT #2” is processed when the culture reaches OD=0.6. C. A representative gel (3% alkaline agarose) of DNA-dU density determination in a 234 nt long fragment of pBR322. D. Quantification of the DNA-dU density (presented as frequency, = 1/density, and in the same scale as in “B”, for direct comparison) in the dut ung thyA mutant grown in the presence of absence of dT for 5 hours, from several gels like in “C”.

Fig. 6.. The effect of rN incorporation and excision on TLD.

A. A scheme of the possible futile DNA-rU misincorporation/excision cycle. B. TLD kinetics of the dinB thyA and umuCD thyA mutants. The strains are: thyA, KKW58; dinB thyA, KJK90; umuCD thyA, KJK87; dinB umuCD thyA, RA36. C. TLD kinetics of the rnhA thyA and rnhB thyA mutants. The strains are: thyA, KKW58; rnhA thyA, RA33; rnhB thyA, RA34; rnhAB thyA, RA35. D. A representative gel (1.1% agarose) of DNA-rN density determination in plasmid DNA (plasmid is pEAK86) isolated from rnhB mutant cells. +dT, growth in the presence of thymidine; –dT, starvation without thymidine. RNase HII, in vitro treatment with RNase HII. Strains are: rnhB, RA34; rnhAB, RA35. E. DNA-rN density (presented as frequency = 1/density) in the rnhB and rnhAB mutants grown in the presence of absence of dT for 5 hours, from several gels like in “E”. F. Evolution of the chromosomal DNA amount in the thyA (KKW58) versus rnhAB thyA (RA35) mutants during dTTP starvation. G. TLD kinetics of the dinB rnhAB thyA and umuCD rnhAB thyA mutants. The strains are: rnhAB thyA, RA35; umuCD rnhAB thyA, RA40; dinB rnhAB thyA, RA39; dinB umuCD rnhAB thyA, RA41.

Fig. 7.. Accumulation of ss-gaps during T-starvation.

A. Evolution of the chromosomal DNA amount in the thyA (KKW58) versus recF thyA (RA31) mutants during T-starvation. B. An example of plug-blot procedure to quantify ssDNA accumulation during T-starvation. After electric transfer to positively-charged nylon membrane, the genomic DNA was hybridized to the total genomic probe. C. Accumulation of ssDNA in percentage to the total DNA signal in the WT cell (AB1157) cultures grown in the presence of the indicated concentrations of AZT. D. The level of ssDNA in thyA mutant (KKW58) cultures grown in the presence (+dT) or absence (–dT) of thymidine. E. The level of ssDNA in the thyA recF (RA31) mutant. F. The level of ssDNA in the thyA dut ung (RA18) mutant.

Fig. 8.. Persistent single-strand gaps kill.

A. TLD kinetics of the thyA mutant (KKW58) in the presence of AZT. After growth in the medium +dT, cells were washed and resuspended in the same volume of the same medium, but without dT, supplemented or not with 100 ng/ml AZT. The ThyA+ strain (KKW59) is also shown, to demonstrate the toxicity of this AZT dose in this medium (without dT). B. TLD kinetics of the thyA recF mutant (RA31) in the presence of 100 ng/ml AZT (done like in “A”). The ThyA+ recF strain (RA48) is also shown, to illustrate AZT toxicity. C. A model to explain loss of the replication forks due to ss-gaps accumulating outside the “safety zone” (green rectangle) around the replication points, within which DNA with ss-interruptions is safe (note the Okazaki fragments on the lagging strand). Red arrowheads mark the position of double-strand breaks. D. The known and the suspected unknown aspects of TLD. The two known aspects, instability of ss-gaps and SOS-induction, are linked with replication forks. However TLD in the thyA dut ung mutant suggests an unknown, replication-independent pathway.