Bacterial nucleoid is a riddle wrapped in a mystery inside an enigma.

Bacterial chromosome, the nucleoid, is traditionally modeled as a rosette of DNA mega-loops, organized around proteinaceous central scaffold by nucleoid-associated proteins (NAPs), and mixed with the cytoplasm by transcription and translation. Electron microscopy of fixed cells confirms dispersal of the cloud-like nucleoid within the ribosome-filled cytoplasm. Here, I discuss evidence that the nucleoid in live cells forms DNA phase separate from riboprotein phase, the "riboid." I argue that the nucleoid-riboid interphase, where DNA interacts with NAPs, transcribing RNA polymerases, nascent transcripts, and ssRNA chaperones, forms the transcription zone. An active part of phase separation, transcription zone enforces segregation of the centrally positioned information phase (the nucleoid) from the surrounding action phase (the riboid), where translation happens, protein accumulates, and metabolism occurs. I speculate that HU NAP mostly tiles up the nucleoid periphery-facilitating DNA mobility but also supporting transcription in the interphase. Besides extruding plectonemically supercoiled DNA mega-loops, condensins could compact them into solenoids of uniform rings, while HU could support rigidity and rotation of these DNA rings. The two-phase cytoplasm arrangement allows the bacterial cell to organize the central dogma activities, where (from the cell center to its periphery) DNA replicates and segregates, DNA is transcribed, nascent mRNA is handed over to ribosomes, mRNA is translated into proteins, and finally, the used mRNA is recycled into nucleotides at the inner membrane. The resulting information-action conveyor, with one activity naturally leading to the next one, explains the efficiency of prokaryotic cell design-even though its main intracellular transportation mode is free diffusion.

Degron-Controlled Protein Degradation in Escherichia coli: New Approaches and Parameters.

Protein degron tags have proven to be uniquely useful for the characterization of gene function. Degrons can mediate quick depletion, usually within minutes, of a protein of interest, allowing researchers to characterize cellular responses to the loss of function. To develop a general-purpose degron tool in Escherichia coli, we sought to build upon a previously characterized system of SspB-dependent inducible protein degradation. For this, we created a family of expression vectors containing a destabilized allele of SspB, capable of a rapid and nearly perfect "off-to-on" induction response. Using this system, we demonstrated excellent control over several DNA metabolism enzymes. However, other substrates did not respond to degron tagging in such an ideal manner, indicating the apparent limitations of SspB-dependent systems. Several degron-tagged proteins were degraded too slowly to be completely depleted during active growth, whereas others appeared to be completely refractory to degron-promoted degradation. Thus, only a minority of our, admittedly biased, selection of degron substrates proved to be amenable to efficient SspB-catalyzed degradation. We also uncovered an apparent stalling and/or disengagement of ClpXP from a degron-tagged allele of beta-galactosidase (beta-gal). While a degron-containing fusion peptide attached to the carboxy-terminus of beta-gal was degraded quantitatively, no reductions in beta-gal activity or concentration were detected, demonstrating an apparently novel mechanism of protease resistance. We conclude that substrate-dependent effects of the SspB system present a continued challenge to the widespread adoption of this degron system. For substrates that prove to be degradable, we provide a series of titratable SspB-expression vehicles.

Degron-controlled protein degradation in Escherichia coli: New Approaches and Parameters.

Protein degron tags have proven uniquely useful for characterization of gene function. Degrons mediate quick depletion, usually within minutes, of a protein of interest - allowing researchers to characterize cellular responses to the loss of function. To develop a general purpose degron tool in E. coli, we sought to build upon a previously characterized system of SspB-dependent inducible protein degradation. For this, we created a family of expression vectors containing a destabilized allele of SspB, capable of a rapid and nearly perfect "off-to-on" induction response. Using this system, we demonstrated control over several enzymes of DNA metabolism, but also found with other substates apparent limitations of a SspB-dependent system. Several degron target proteins were degraded too slowly to affect their complete depletion during active growth, whereas others appeared completely refractory to degron-promoted degradation. We demonstrated that a model substrate, beta-galactosidase, was positively recognized as a degron substrate, but failed to be degraded by the ClpXP protease - demonstrating an apparently unknown mechanism of protease resistance. Thus, only a minority of our, admittedly biased, selection of degron substates proved amenable to rapid SspB-catalyzed degradation. We conclude that substrate-dependence of the SspB system remains a critical factor for the success of this degron system. For substrates that prove degradable, we provide a series of titratable SspB-expression vehicles.

Synthetic lethal mutants in Escherichia coli define pathways necessary for survival with RNase H deficiency.

Ribonucleotides frequently contaminate DNA and, if not removed, cause genomic instability. Consequently, all organisms are equipped with RNase H enzymes to remove RNA-DNA hybrids (RDHs). Escherichia coli lacking RNase HI (rnhA) and RNase HII (rnhB) enzymes, the ∆rnhA ∆rnhB double mutant, accumulates RDHs in its DNA. These RDHs can convert into RNA-containing DNA lesions (R-lesions) of unclear nature that compromise genomic stability. The ∆rnhAB double mutant has severe phenotypes, like growth inhibition, replication stress, sensitivity to ultraviolet radiation, SOS induction, increased chromosomal fragmentation, and defects in nucleoid organization. In this study, we found that RNase HI deficiency also alters wild-type levels of DNA supercoiling. Despite these severe chromosomal complications, ∆rnhAB double mutant survives, suggesting that dedicated pathways operate to avoid or repair R-lesions. To identify these pathways, we systematically searched for mutants synthetic lethal (colethal) with the rnhAB defect using an unbiased color screen and a candidate gene approach. We identified both novel and previously reported rnhAB-colethal and -coinhibited mutants, characterized them, and sorted them into avoidance or repair pathways. These mutants operate in various parts of nucleic acid metabolism, including replication fork progression, R-loop prevention and removal, nucleoid organization, tRNA modification, recombinational repair, and chromosome-dimer resolution, demonstrating the pleiotropic nature of RNase H deficiency. IMPORTANCE Ribonucleotides (rNs) are structurally very similar to deoxyribonucleotides. Consequently, rN contamination of DNA is common and pervasive across all domains of life. Failure to remove rNs from DNA has severe consequences, and all organisms are equipped with RNase H enzymes to remove RNA-DNA hybrids. RNase H deficiency leads to complications in bacteria, yeast, and mouse, and diseases like progressive external ophthalmoplegia (mitochondrial defects in RNASEH1) and Aicardi-Goutières syndrome (defects in RNASEH2) in humans. Escherichia coli ∆rnhAB mutant, deficient in RNases H, has severe chromosomal complications. Despite substantial problems, nearly half of the mutant population survives. We have identified novel and previously confirmed pathways in various parts of nucleic acid metabolism that ensure survival with RNase H deficiency.

Catastrophic chromosome fragmentation probes the nucleoid structure and dynamics in Escherichia coli.

Escherichia coli cells treated with a combination of cyanide (CN) and hydrogen peroxide (HP) succumb to catastrophic chromosome fragmentation (CCF), detectable in pulsed-field gels as >100 double-strand breaks per genome equivalent. Here we show that CN + HP-induced double-strand breaks are independent of replication and occur uniformly over the chromosome,-therefore we used CCF to probe the nucleoid structure by measuring DNA release from precipitated nucleoids. CCF releases surprisingly little chromosomal DNA from the nucleoid suggesting that: (i) the nucleoid is a single DNA-protein complex with only limited stretches of protein-free DNA and (ii) CN + HP-induced breaks happen within these unsecured DNA stretches, rather than at DNA attachments to the central scaffold. Mutants lacking individual nucleoid-associated proteins (NAPs) release more DNA during CCF, consistent with NAPs anchoring chromosome to the central scaffold (Dps also reduces the number of double-strand breaks directly). Finally, significantly more broken DNA is released once ATP production is restored, with about two-thirds of this ATP-dependent DNA release being due to transcription, suggesting that transcription complexes act as pulleys to move DNA loops. In addition to NAPs, recombinational repair of double-strand breaks also inhibits DNA release by CCF, contributing to a dynamic and complex nucleoid structure.

Robust linear DNA degradation supports replication-initiation-defective mutants in Escherichia coli.

RecBCD helicase/nuclease supports replication fork progress via recombinational repair or linear DNA degradation, explaining recBC mutant synthetic lethality with replication elongation defects. Since replication initiation defects leave chromosomes without replication forks, these should be insensitive to the recBCD status. Surprisingly, we found that both Escherichia coli dnaA46(Ts) and dnaC2(Ts) initiation mutants at semi-permissive temperatures are also recBC-colethal. Interestingly, dnaA46 recBC lethality suppressors suggest underinitiation as the problem, while dnaC2 recBC suppressors signal overintiation. Using genetic and physical approaches, we studied the dnaA46 recBC synthetic lethality, for the possibility that RecBCD participates in replication initiation. Overproduced DnaA46 mutant protein interferes with growth of dnaA+ cells, while the residual viability of the dnaA46 recBC mutant depends on the auxiliary replicative helicase Rep, suggesting replication fork inhibition by the DnaA46 mutant protein. The dnaA46 mutant depends on linear DNA degradation by RecBCD, rather than on recombinational repair. At the same time, the dnaA46 defect also interacts with Holliday junction-moving defects, suggesting reversal of inhibited forks. However, in contrast to all known recBC-colethals, which fragment their chromosomes, the dnaA46 recBC mutant develops no chromosome fragmentation, indicating that its inhibited replication forks are stable. Physical measurements confirm replication inhibition in the dnaA46 mutant shifted to semi-permissive temperatures, both at the level of elongation and initiation, while RecBCD gradually restores elongation and then initiation. We propose that RecBCD-catalyzed resetting of inhibited replication forks allows replication to displace the "sticky" DnaA46(Ts) protein from the chromosomal DNA, mustering enough DnaA for new initiations.

Thymine-starvation-induced chromosomal fragmentation is not required for thymineless death in Escherichia coli.

Thymine or thymidine starvation induces robust chromosomal fragmentation in Escherichia coli thyA deoCABD mutants and is proposed to be the cause of thymineless death (TLD). However, fragmentation kinetics challenges the idea that fragmentation causes TLD, by peaking before the onset of TLD and disappearing by the time TLD accelerates. Quantity and kinetics of fragmentation also stay unchanged in hyper-TLD-exhibiting recBCD mutant, making its faster and deeper TLD independent of fragmentation as well. Elimination of fragmentation without affecting cellular metabolism did not abolish TLD in the thyA mutant, but reduced early TLD in the thyA recBCD mutant, suggesting replication-dependent, but undetectable by pulsed-field gel, double-strand breaks contributed to TLD. Chromosomal fragmentation, but not TLD, was eliminated in both the thyA and thyA recBCD mutants harboring deoCABD operon. The expression of a single gene, deoA, encoding thymidine phosphorylase, was sufficient to abolish fragmentation, suggesting thymidine-to-thymine interconversion during T-starvation being a key factor. Overall, this study reveals that chromosomal fragmentation, a direct consequence of T-starvation, is either dispensable or redundant for the overall TLD pathology, including hyper-TLD in the recBCD mutant. Replication forks, unlike chromosomal fragmentation, may provide a minor contribution to TLD, but only in the repair-deficient thyA deoCABD recBCD mutant.

Nitric oxide precipitates catastrophic chromosome fragmentation by bolstering both hydrogen peroxide and Fe(II) Fenton reactants in E. coli.

Immune cells kill invading microbes by producing reactive oxygen and nitrogen species, primarily hydrogen peroxide (H2O2) and nitric oxide (NO). We previously found that NO inhibits catalases in Escherichia coli, stabilizing H2O2 around treated cells and promoting catastrophic chromosome fragmentation via continuous Fenton reactions generating hydroxyl radicals. Indeed, H2O2-alone treatment kills catalase-deficient (katEG) mutants similar to H2O2+NO treatment. However, the Fenton reaction, in addition to H2O2, requires Fe(II), which H2O2 excess instantly converts into Fenton-inert Fe(III). For continuous Fenton when H2O2 is stable, a supply of reduced iron becomes necessary. We show here that this supply is ensured by Fe(II) recruitment from ferritins and Fe(III) reduction by flavin reductase. Our observations also concur with NO-mediated respiration inhibition that drives Fe(III) reduction. We modeled this NO-mediated inhibition via inactivation of ndh and nuo respiratory enzymes responsible for the step of NADH oxidation, which results in increased NADH pools driving flavin reduction. We found that, like the katEG mutant, the ndh nuo double mutant is similarly sensitive to H2O2-alone and H2O2+NO treatments. Moreover, the quadruple katEG ndh nuo mutant lacking both catalases and efficient respiration was rapidly killed by H2O2-alone, but this killing was delayed by NO, rather than potentiated by it. Taken together, we conclude that NO boosts the levels of both H2O2 and Fe(II) Fenton reactants, making continuous hydroxyl-radical production feasible and resulting in irreparable oxidative damage to the chromosome.

Oxidative Damage Blocks Thymineless Death and Trimethoprim Poisoning in Escherichia coli.

Cells that cannot synthesize one of the DNA precursors, dTTP, due to thyA mutation or metabolic poisoning, undergo thymineless death (TLD), a chromosome-based phenomenon of unclear mechanisms. In Escherichia coli, thymineless death is caused either by denying thyA mutants thymidine supplementation or by treating wild-type cells with trimethoprim. Two recent reports promised a potential breakthrough in TLD understanding, suggesting significant oxidative damage during thymine starvation. Oxidative damage in vivo comes from Fenton's reaction when hydrogen peroxide meets ferrous iron to produce hydroxyl radical. Therefore, TLD could kill via irreparable double-strand breaks behind replication forks when starvation-caused single-strand DNA gaps are attacked by hydroxyl radicals. We tested the proposed Fenton-TLD connection in both thyA mutants denied thymidine, as well as in trimethoprim-treated wild-type (WT) cells, under the following three conditions: (i) intracellular iron chelation, (ii) mutational inactivation of hydrogen peroxide (HP) scavenging, and (iii) acute treatment with sublethal HP concentrations. We found that TLD kinetics are affected by neither iron chelation nor HP stabilization in cultures, indicating no induction of oxidative damage during thymine starvation. Moreover, acute exogenous HP treatments completely block TLD, apparently by blocking cell division, which may be a novel TLD prerequisite. Separately, the acute trimethoprim sensitivity of the rffC and recBCD mutants demonstrates how bactericidal power of this antibiotic could be amplified by inhibiting the corresponding enzymes. IMPORTANCE Mysterious thymineless death strikes cells that are starved for thymine and therefore replicating their chromosomal DNA without dTTP. After 67 years of experiments testing various obvious and not so obvious explanations, thymineless death is still without a mechanism. Recently, oxidative damage via in vivo Fenton's reaction was proposed as a critical contributor to the irreparable chromosome damage during thymine starvation. We have tested this idea by either blocking in vivo Fenton's reaction (expecting no thymineless death) or by amplifying oxidative damage (expecting hyperthymineless death). Instead, we found that blocking Fenton's reaction has no influence on thymineless death, while amplifying oxidative damage prevents thymineless death altogether. Thus, oxidative damage does not contribute to thymineless death, while the latter remains enigmatic.

Electron Microscopy Reveals Unexpected Cytoplasm and Envelope Changes during Thymineless Death in Escherichia coli.

Bacterial rod-shaped cells experiencing irreparable chromosome damage should filament without other morphological changes. Thymineless death (TLD) strikes thymidine auxotrophs denied external thymine/thymidine (T) supplementation. Such T-starved cells cannot produce the DNA precursor dTTP and therefore stop DNA replication. Stalled replication forks in T-starved cells were always assumed to experience mysterious chromosome lesions, but TLD was recently found to happen even without origin-dependent DNA replication, with the chromosome still remaining the main TLD target. T starvation also induces morphological changes, as if thymidine prevents cell envelope or cytoplasm problems that otherwise translate into chromosome damage. Here, we used transmission electron microscopy (TEM) to examine cytoplasm and envelope changes in T-starved Escherichia coli cells, using treatment with a DNA gyrase inhibitor as a control for "pure" chromosome death. Besides the expected cell filamentation in response to both treatments, we see the following morphological changes specific for T starvation and which might lead to chromosome damage: (i) significant cell widening, (ii) nucleoid diffusion, (iii) cell pole damage, and (iv) formation of numerous cytoplasmic bubbles. We conclude that T starvation does impact both the cytoplasm and the cell envelope in ways that could potentially affect the chromosome. IMPORTANCE Thymineless death is a dramatic and medically important phenomenon, the mechanisms of which remain a mystery. Unlike most other auxotrophs in the absence of the required supplement, thymidine-requiring E. coli mutants not only go static in the absence of thymidine, but rapidly die of chromosomal damage of unclear nature. Since this chromosomal damage is independent of replication, we examined fine morphological changes in cells undergoing thymineless death in order to identify what could potentially affect the chromosome. Here, we report several cytoplasm and cell envelope changes that develop in thymidine-starved cells but not in gyrase inhibitor-treated cells (negative control) that could be linked to subsequent irreparable chromosome damage. This is the first electron microscopy study of cells undergoing "genetic death" due to irreparable chromosome lesions.

Catalase inhibition by nitric oxide potentiates hydrogen peroxide to trigger catastrophic chromosome fragmentation in Escherichia coli.

Hydrogen peroxide (H2O2, HP) is a universal toxin that organisms deploy to kill competing or invading cells. Bactericidal action of H2O2 presents several questions. First, the lethal H2O2 concentrations in bacterial cultures are 1000x higher than, for example, those calculated for the phagosome. Second, H2O2-alone kills bacteria in cultures either by mode-one, via iron-mediated chromosomal damage, or by mode-two, via unknown targets, but the killing mode in phagosomes is unclear. Third, phagosomal H2O2 toxicity is enhanced by production of nitric oxide (NO), but in vitro studies disagree: some show NO synergy with H2O2 antimicrobial action, others instead report alleviation. To investigate this "NO paradox," we treated Escherichia coli with various concentrations of H2O2-alone or H2O2+NO, measuring survival and chromosome stability. We found that all NO concentrations make sublethal H2O2 treatments highly lethal, via triggering catastrophic chromosome fragmentation (mode-one killing). Yet, NO-alone is not lethal, potentiating H2O2 toxicity by blocking H2O2 scavenging in cultures. Catalases represent obvious targets of NO inhibition, and catalase-deficient mutants are indeed killed equally by H2O2-alone or H2O2+NO treatments, also showing similar levels of chromosome fragmentation. Interestingly, iron chelation blocks chromosome fragmentation in catalase-deficient mutants without blocking H2O2-alone lethality, indicating mode-two killing. In fact, mode-two killing of WT cells by much higher H2O2 concentrations is transiently alleviated by NO, reproducing the "NO paradox." We conclude that NO potentiates H2O2 toxicity by promoting mode-one killing (via catastrophic chromosome fragmentation) by otherwise static low H2O2 concentrations, while transiently suppressing mode-two killing by immediately lethal high H2O2 concentrations.

Ultraviolet-induced RNA:DNA hybrids interfere with chromosomal DNA synthesis.

Ultraviolet (UV) induces pyrimidine dimers (PDs) in DNA and replication-dependent fragmentation in chromosomes. The rnhAB mutants in Escherichia coli, accumulating R-loops and single DNA-rNs, are generally resistant to DNA damage, but are surprisingly UV-sensitive, even though they remove PDs normally, suggesting irreparable chromosome lesions. We show here that the RNase H defect does not cause additional chromosome fragmentation after UV, but inhibits DNA synthesis after replication restart. Genetic analysis implies formation of R-loop-anchored transcription elongation complexes (R-loop-aTECs) in UV-irradiated rnhAB mutants, predicting that their chromosomal DNA will accumulate: (i) RNA:DNA hybrids; (ii) a few slow-to-remove PDs. We confirm both features and also find that both, surprisingly, depend on replication restart. Finally, enriching for the UV-induced RNA:DNA hybrids in the rnhAB uvrA mutants also co-enriches for PDs, showing their co-residence in the same structures. We propose that PD-triggered R-loop-aTECs block head-on replication in RNase H-deficient mutants.

Exopolysaccharide defects cause hyper-thymineless death in Escherichia coli via massive loss of chromosomal DNA and cell lysis.

Thymineless death in Escherichia coli thyA mutants growing in the absence of thymidine (dT) is preceded by a substantial resistance phase, during which the culture titer remains static, as if the chromosome has to accumulate damage before ultimately failing. Significant chromosomal replication and fragmentation during the resistance phase could provide appropriate sources of this damage. Alternatively, the initial chromosomal replication in thymine (T)-starved cells could reflect a considerable endogenous dT source, making the resistance phase a delay of acute starvation, rather than an integral part of thymineless death. Here we identify such a low-molecular-weight (LMW)-dT source as mostly dTDP-glucose and its derivatives, used to synthesize enterobacterial common antigen (ECA). The thyA mutant, in which dTDP-glucose production is blocked by the rfbA rffH mutations, lacks a LMW-dT pool, the initial DNA synthesis during T-starvation and the resistance phase. Remarkably, the thyA mutant that makes dTDP-glucose and initiates ECA synthesis normally yet cannot complete it due to the rffC defect, maintains a regular LMW-dT pool, but cannot recover dTTP from it, and thus suffers T-hyperstarvation, dying precipitously, completely losing chromosomal DNA and eventually lysing, even without chromosomal replication. At the same time, its ECA+thyA parent does not lyse during T-starvation, while both the dramatic killing and chromosomal DNA loss in the ECA-deficient thyA mutants precede cell lysis. We conclude that: 1) the significant pool of dTDP-hexoses delays acute T-starvation; 2) T-starvation destabilizes even nonreplicating chromosomes, while T-hyperstarvation destroys them; and 3) beyond the chromosome, T-hyperstarvation also destabilizes the cell envelope.

Half-Intercalation Stabilizes Slipped Mispairing and Explains Genome Vulnerability to Frameshift Mutagenesis by Endogenous "Molecular Bookmarks".

Some 60 years ago chemicals that intercalate between base pairs of duplex DNA were found to amplify frameshift mutagenesis. Surprisingly, the robust induction of frameshifts by intercalators still lacks a mechanistic model, leaving this classic phenomenon annoyingly intractable. A promising idea of asymmetric half-intercalation-stabilizing frameshift intermediates during DNA synthesis has never been developed into a model. Instead, researchers of frameshift mutagenesis embraced the powerful slipped-mispairing concept that unexpectedly struggled with the role of intercalators in frameshifting. It is proposed that the slipped mispairing and the half-intercalation ideas are two sides of the same coin. Further, existing findings are reviewed to test predictions of the combined "half-intercalation into the slipped-mispairing intermediate" model against accumulated knowledge. The existence of potential endogenous intercalators and the phenomenon of "DNA bookmarks" reveal ample possibilities for natural frameshift mutagenisis in the cell. From this alarming perspective, it is discussed how the cell could prevent genome deterioration from frameshift mutagenesis.

Thymineless Death in Escherichia coli Is Unaffected by Chromosomal Replication Complexity.

Thymineless death (TLD) is a rapid loss of viability of unclear mechanism in cultures of thyA mutants starved for thymine/thymidine (T starvation). It is accepted that T starvation repeatedly breaks replication forks, while recombinational repair restores them, but when the resulting futile breakage-repair cycle affects the small replication bubbles at oriC, the origin is degraded, killing the cell. Indeed, cells with increased chromosomal replication complexity (CRC), expressed as an elevated origin/terminus (ori/ter) ratio, die more extensively during TLD. Here we tested this logic by elevating the CRC in Escherichia colithyA mutants before T starvation, anticipating exaggerated TLD. Unexpectedly, TLD remained unaffected by a CRC increase to either the natural limit (ori/ter ratio, ∼6) or the functional limit (ori/ter ratio, ∼16). Moreover, when we forced the CRC over the functional limit (ori/ter ratio, ∼30), TLD lessened. Thus, prior overinitiation does not sensitize cells to TLD. In contradiction with the published results, even blocking new replication initiations by the dnaA(Ts) defect at 42°C fails to prevent TLD. Using the thyA dnaA(Ts) mutant in a new T starvation protocol that excludes new initiations, we show that at 42°C, the same degree of TLD still occurs when chromosomes are demonstrably nonreplicating. Remarkably, 80% of the chromosomal DNA in these nonreplicating T-starved cells is still lost, by an unclear mechanism.IMPORTANCE Thymineless death kills cells of any type and is used in anticancer and antimicrobial treatments. We tested the idea that the more replication forks there are in the chromosome during growth, the more extensive the resulting thymineless death. We varied the number of replication forks in the Escherichia coli chromosome, as measured by the origin-to-terminus ratio, ranging it from the normal 2 to 60, and even completely eliminated replication forks in the nonreplicating chromosomes (ori/ter ratio = 1). Unexpectedly, we found that thymineless death is unaffected by the intensity of replication or by its complete absence; we also found that even nonreplicating chromosomes still disappear during thymine starvation. We conclude that thymineless death can kill E. coli independently of chromosomal replication.

Near-continuously synthesized leading strands in Escherichia coli are broken by ribonucleotide excision.

In vitro, purified replisomes drive model replication forks to synthesize continuous leading strands, even without ligase, supporting the semidiscontinuous model of DNA replication. However, nascent replication intermediates isolated from ligase-deficient Escherichia coli comprise only short (on average 1.2-kb) Okazaki fragments. It was long suspected that cells replicate their chromosomal DNA by the semidiscontinuous mode observed in vitro but that, in vivo, the nascent leading strand was artifactually fragmented postsynthesis by excision repair. Here, using high-resolution separation of pulse-labeled replication intermediates coupled with strand-specific hybridization, we show that excision-proficient E. coli generates leading-strand intermediates >10-fold longer than lagging-strand Okazaki fragments. Inactivation of DNA-repair activities, including ribonucleotide excision, further increased nascent leading-strand size to ∼80 kb, while lagging-strand Okazaki fragments remained unaffected. We conclude that in vivo, repriming occurs ∼70× less frequently on the leading versus lagging strands, and that DNA replication in E. coli is effectively semidiscontinuous.

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways.

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

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

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.

When DNA Topology Turns Deadly - RNA Polymerases Dig in Their R-Loops to Stand Their Ground: New Positive and Negative (Super)Twists in the Replication-Transcription Conflict.

Head-on replication-transcription conflict is especially bitter in bacterial chromosomes, explaining why actively transcribed genes are always co-oriented with replication. The mechanism of this conflict remains unclear, besides the anticipated accumulation of positive supercoils between head-on-conflicting polymerases. Unexpectedly, experiments in bacterial and human cells reveal that head-on replication-transcription conflict induces R-loops, indicating hypernegative supercoiling [(-)sc] in the region - precisely the opposite of that assumed. Further, as a result of these R-loops, both replication and transcription in the affected region permanently stall, so the failure of R-loop removal in RNase H-deficient bacteria becomes lethal. How hyper(-)sc emerges in the middle of a positively supercoiled chromosomal domain is a mystery that requires rethinking of topoisomerase action around polymerases.

Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome.

The nucleoid of Escherichia coli comprises DNA, nucleoid associated proteins (NAPs) and RNA, whose role is unclear. We found that lysing bacterial cells embedded in agarose plugs in the presence of RNases caused massive fragmentation of the chromosomal DNA. This RNase-induced chromosomal fragmentation (RiCF) was completely dependent on the presence of RNase around lysing cells, while the maximal chromosomal breakage required fast cell lysis. Cell lysis in plugs without RNAse made the chromosomal DNA resistant to subsequent RNAse treatment. RiCF was not influenced by changes in the DNA supercoiling, but was influenced by growth temperature or age of the culture. RiCF was partially dependent on H-NS, histone-like nucleoid structuring- and global transcription regulator protein. The hupAB deletion of heat-unstable nucleoid protein (HU) caused increase in spontaneous fragmentation that was further increased when combined with deletions in two non-coding RNAs, nc1 and nc5. RiCF was completely dependent upon endonuclease I, a periplasmic deoxyribonuclease that is normally found inhibited by cellular RNA. Unlike RiCF, the spontaneous fragmentation in hupAB nc1 nc5 quadruple mutant was resistant to deletion of endonuclease I. RiCF-like phenomenon was observed without addition of RNase to agarose plugs if EDTA was significantly reduced during cell lysis. Addition of RNase under this condition was synergistic, breaking chromosomes into pieces too small to be retained by the pulsed field gels. RNase-independent fragmentation was qualitatively and quantitatively comparable to RiCF and was partially mediated by endonuclease I.