How To Repair The Damage Tld

doi: ten.1016/j.dnarep.2019.01.002. Epub 2022 Jan 16.

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

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• PMID: 30684682
• PMCID: PMC6382538
• DOI: 10.1016/j.dnarep.2019.01.002

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

T Five Pritha Rao  et al. DNA Repair (Amst). 2019 Mar .

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Abstract

Thymine deprivation in thyA mutant E. coli causes thymineless death (TLD) and is the mode of activeness of popular antibacterial and anticancer drugs, notwithstanding the mechanisms of TLD are notwithstanding unclear. TLD comprises 3 divers phases: resistance, rapid exponential death (RED) and survival, with the nature of the resistance phase and of the transition to the RED phase holding central to TLD pathology. We advise 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 Ruddy phase, eventually rendering the chromosome not-functional. Two obvious sources of the endogenous thymine are deposition of broken chromosomal Dna and recruitment of thymine from stable RNA. Nevertheless, mutants that cannot dethrone broken chromosomal Dna or lack ribo-thymine, instead of shortening the resistance stage, deepen the RED phase, meaning that only a small fraction of T-starved cells tap into these sources. Interestingly, the substantial chromosomal Deoxyribonucleic acid aggregating 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 Deoxyribonucleic acid-dU incorporation and no DNA-dU excision, replication is in fact rescued fifty-fifty 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 fifty-fifty 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.

Copyright © 2022 Elsevier B.V. All rights reserved.

Figures

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 blackness, compounds of RNA metabolism are in orange. Genes are colored co-ordinate to functions: general (blue), dTTP-synthesis (regal) or Dna repair (magenta). The large arrow shades are: green, general biosynthesis; yellow, save; blue, DNA degradation; cyan, RNA deposition; magenta, futile DNA-dU misincorporation/excision bicycle; orange, futile Deoxyribonucleic acid-rU misincorporation/excision bicycle. B. Extended fourth dimension course of the civilisation titer during thymine starvation in our standard atmospheric condition, to highlight the survival phase. The strains are: ThyA+, KKW59; thyA, KKW58. The three phases of TLD are shown in colour: green for the resistance phase, ruby for the RED phase, purple for the survival stage. 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, too as the aforementioned 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. Diverse models of replisomes traversing A-runs in the template Dna during T-starvation that nosotros 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 five) ± 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 agile (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/one thousand². F. Time form of TLD in the recBCD sbcBC thyA mutant. The strains are: thyA, KKW58; recBCD sbcBC thyA, RA45. Grand. Change in the total amount of chromosomal Dna over time ("chromosomal Deoxyribonucleic acid evolution") in the thyA mutant (KKW58) during T-starvation. The first 2 phases of T-starvation are shown in the same colors every bit in Fig. 1A. H. Expectations about the origin and terminus re-create number evolution during T-starvation according to the intrachromosomal DNA redistribution thought. I. Evolution of the corporeality of origin and terminus nether the same weather condition equally in "Thou".

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

A. A scheme of stable RNAs (tRNA has 1 rT, 23S rRNA has two) and how many thymine residues they can yield per average chop-chop-growing cell. B. Fourth dimension course of TLD after 15' @ 54°C rut daze. 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 respective RNA species in the cells incubated without dT to cells from the aforementioned culture incubated in the presence of dT. The bodily measurements, normalized to time 0, are shown in Figures S3B and S3C. D. Recruitment of rT by degrading stable RNA kills 2 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. Fourth dimension course of TLD in the thyA trmA mutant (RA9). F. Fourth dimension course of TLD in the thyA rumA rumB mutant (RA13). Thou. 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. Development of the chromosomal Deoxyribonucleic acid absolute amount in the thyA and trmA thyA mutants (strains like in "East") during T-starvation.

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

A. A scheme of Deoxyribonucleic acid-dU misincorporation and its outcome in thyA, ung thyA, dut thyA and dut ung thyA mutants undergoing thymine starvation. Green fill, growth with dT. Light pinkish fill, attempted replication without dT using little dU (Dut+) with active DNA-uracil removal (Ung+). Xanthous fill up, replication without dT using piffling dU (Dut+), with no Deoxyribonucleic acid-uracil removal (ung). Pink make full, attempted replication without dT just with plenty of dU (dut) with active DNA-uracil removal (Ung+). Lite green fill up, 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 Deoxyribonucleic acid-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 (ane.1% agarose) of DNA-dU density determination in plasmid DNA (pMTL20). UDG, treatment with uracil-DNA glycosylase; Exo III, treatment with exonuclease 3; U + East, handling with both UDG and Exo 3. B. Quantification of the Deoxyribonucleic acid-dU density (presented as frequency = ane/density) in the ung thyA mutant grown in the presence of absence of dT, from several gels similar in "A". The "–dT" civilisation is processed 5 hours after dT removal by filtration. At that place are two dissimilar conditions of growth in the presence of dT, though: "+dT #1" is too candy five hours later on the filtration (but dT was re-added in this instance), so the culture becomes stationary. In dissimilarity, "+dT #2" is processed when the civilization reaches OD=0.6. C. A representative gel (3% alkaline agarose) of Deoxyribonucleic acid-dU density decision in a 234 nt long fragment of pBR322. D. Quantification of the Dna-dU density (presented every bit frequency, = one/density, and in the same calibration as in "B", for directly comparison) in the dut ung thyA mutant grown in the presence of absenteeism 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 bicycle. 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.i% 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 handling with RNase HII. Strains are: rnhB, RA34; rnhAB, RA35. E. Deoxyribonucleic acid-rN density (presented as frequency = 1/density) in the rnhB and rnhAB mutants grown in the presence of absence of dT for five hours, from several gels like in "East". F. Evolution of the chromosomal Deoxyribonucleic acid 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. seven.. 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 instance of plug-blot procedure to quantify ssDNA aggregating during T-starvation. After electrical transfer to positively-charged nylon membrane, the genomic Dna was hybridized to the total genomic probe. C. Aggregating 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. Eastward. 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 book 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) effectually the replication points, within which Dna with ss-interruptions is safe (note the Okazaki fragments on the lagging strand). Red arrowheads marker the position of double-strand breaks. D. The known and the suspected unknown aspects of TLD. The 2 known aspects, instability of ss-gaps and SOS-consecration, are linked with replication forks. However TLD in the thyA dut ung mutant suggests an unknown, replication-independent pathway.

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