R-loops and nicks initiate DNA breakage and genome instability in non-growing Escherichia coli.

Double-stranded DNA ends, often from replication, drive genomic instability, yet their origin in non-replicating cells is unknown. Here we show that transcriptional RNA/DNA hybrids (R-loops) generate DNA ends that underlie stress-induced mutation and amplification. Depleting RNA/DNA hybrids with overproduced RNase HI reduces both genomic changes, indicating RNA/DNA hybrids as intermediates in both. An Mfd requirement and inhibition by translation implicate transcriptional R-loops. R-loops promote instability by generating DNA ends, shown by their dispensability when ends are provided by I-SceI endonuclease. Both R-loops and single-stranded endonuclease TraI are required for end formation, visualized as foci of a fluorescent end-binding protein. The data suggest that R-loops prime replication forks that collapse at single-stranded nicks, producing ends that instigate genomic instability. The results illuminate how DNA ends form in non-replicating cells, identify R-loops as the earliest known mutation/amplification intermediate, and suggest that genomic instability during stress could be targeted to transcribed regions, accelerating adaptation.

Gross chromosomal rearrangement mediated by DNA replication in stressed cells: evidence from Escherichia coli.

Gross chromosomal rearrangements (GCRs), or changes in chromosome structure, play central roles in evolution and are central to cancer formation and progression. GCRs underlie copy number variation (CNV), and therefore genomic disorders that stem from CNV. We study amplification in Escherichia coli as a model system to understand mechanisms and circumstances of GCR formation. Here, we summarize observations that led us to postulate that GCR occurs by a replicative mechanism as part of activated stress responses. We report that we do not find RecA to be downregulated by stress on a population basis and that constitutive expression of RecA does not inhibit amplification, as would be expected if downregulation of RecA made cells permissive for nonhomologous recombination. Strains deleted for the genes for three proteins that inhibit RecA activity, psiB, dinI, and recX, all show unaltered amplification, suggesting that if they do downregulate RecA indirectly, this activity does not promote amplification.

Global chromosomal structural instability in a subpopulation of starving Escherichia coli cells.

Copy-number variations (CNVs) constitute very common differences between individual humans and possibly all genomes and may therefore be important fuel for evolution, yet how they form remains elusive. In starving Escherichia coli, gene amplification is induced by stress, controlled by the general stress response. Amplification has been detected only encompassing genes that confer a growth advantage when amplified. We studied the structure of stress-induced gene amplification in starving cells in the Lac assay in Escherichia coli by array comparative genomic hybridization (aCGH), with polymerase chain reaction (pcr) and DNA sequencing to establish the structures generated. About 10% of 300 amplified isolates carried other chromosomal structural change in addition to amplification. Most of these were inversions and duplications associated with the amplification event. This complexity supports a mechanism similar to that seen in human non-recurrent copy number variants. We interpret these complex events in terms of repeated template switching during DNA replication. Importantly, we found a significant occurrence (6 out of 300) of chromosomal structural changes that were apparently not involved in the amplification event. These secondary changes were absent from 240 samples derived from starved cells not carrying amplification, suggesting that amplification happens in a differentiated subpopulation of stressed cells licensed for global chromosomal structural change and genomic instability. These data imply that chromosomal structural changes occur in bursts or showers of instability that may have the potential to drive rapid evolution.

Separate DNA Pol II- and Pol IV-dependent pathways of stress-induced mutation during double-strand-break repair in Escherichia coli are controlled by RpoS.

Previous work showed that about 85% of stress-induced mutations associated with DNA double-strand break repair in carbon-starved Escherichia coli result from error-prone DNA polymerase IV (Pol IV) (DinB) and that the mutagenesis is controlled by the RpoS stress response, which upregulates dinB. We report that the remaining mutagenesis requires high-fidelity Pol II, and that this component also requires RpoS. The results identify a second DNA polymerase contributing to stress-induced mutagenesis and show that RpoS promotes mutagenesis by more than the simple upregulation of dinB.

Competition of Escherichia coli DNA polymerases I, II and III with DNA Pol IV in stressed cells.

Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at approximately 200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB approximately 10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB) repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s), tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also provides a sensitive indicator for DNA polymerase competition and choice in vivo.

On the mechanism of gene amplification induced under stress in Escherichia coli.

Gene amplification is a collection of processes whereby a DNA segment is reiterated to multiple copies per genome. It is important in carcinogenesis and resistance to chemotherapeutic agents, and can underlie adaptive evolution via increased expression of an amplified gene, evolution of new gene functions, and genome evolution. Though first described in the model organism Escherichia coli in the early 1960s, only scant information on the mechanism(s) of amplification in this system has been obtained, and many models for mechanism(s) were possible. More recently, some gene amplifications in E. coli were shown to be stress-inducible and to confer a selective advantage to cells under stress (adaptive amplifications), potentially accelerating evolution specifically when cells are poorly adapted to their environment. We focus on stress-induced amplification in E. coli and report several findings that indicate a novel molecular mechanism, and we suggest that most amplifications might be stress-induced, not spontaneous. First, as often hypothesized, but not shown previously, certain proteins used for DNA double-strand-break repair and homologous recombination are required for amplification. Second, in contrast with previous models in which homologous recombination between repeated sequences caused duplications that lead to amplification, the amplified DNAs are present in situ as tandem, direct repeats of 7-32 kilobases bordered by only 4 to 15 base pairs of G-rich homology, indicating an initial non-homologous recombination event. Sequences at the rearrangement junctions suggest nonhomologous recombination mechanisms that occur via template switching during DNA replication, but unlike previously described template switching events, these must occur over long distances. Third, we provide evidence that 3'-single-strand DNA ends are intermediates in the process, supporting a template-switching mechanism. Fourth, we provide evidence that lagging-strand templates are involved. Finally, we propose a novel, long-distance template-switching model for the mechanism of adaptive amplification that suggests how stress induces the amplifications. We outline its possible applicability to amplification in humans and other organisms and circumstances.

Patient-led variable dosing with balsalazide as long-term therapy for maintenance in ulcerative colitis: a 3-year prospective observational study.

BACKGROUND: The patient-centred approach is new to the management of ulcerative colitis. To date, it has only been shown to be successful in a short-term study. AIM: To assess the feasibility, safety and efficacy of patient-led dosing using balsalazide in the long-term treatment of ulcerative colitis. METHODS: This was a 3-year, two-cohort, multi-centre study: one cohort was in stable remission (52 patients) and the other was newly in remission (76 patients) from ulcerative colitis. Two 750-mg balsalazide capsules were given twice daily for maintenance, increased by 750-mg increments to a maximum of 6 g for up to 7 days depending on symptom severity. Clinical assessments were made every 12-14 weeks; laboratory assessments were made every 6 months. RESULTS: The average median daily dose of balsalazide was 3 g (range, 1.5-6 g). In the cohort with stable remission, 23 patients (44%) had relapsed by 3 years [median time to relapse, > 1095 days (36 months)]. In the cohort newly in remission, these values were 45 patients (59%) and 656 days (22 months), respectively. In the cohort with stable remission, the time since last relapse was significantly associated with relapse during the first year of treatment (P < 0.033). CONCLUSIONS: Long-term, patient-led, maintenance treatment with balsalazide is well tolerated with a good safety profile and is effective for patients with ulcerative colitis.

A double-blind comparison of balsalazide, 6.75 g daily, and sulfasalazine, 3 g daily, in patients with newly diagnosed or relapsed active ulcerative colitis.

BACKGROUND: Sulfasalazine is well established in the treatment of active ulcerative colitis. Intolerance to sulfasalazine, however, is a common problem. Balsalazide has been designed to deliver 5-aminosalicylic acid to the colon without the poor tolerability of sulfasalazine. AIM: To compare the safety and efficacy of balsalazide, 6.75 g daily, with sulfasalazine, 3 g daily, in the treatment of active ulcerative colitis of all grades of severity. METHODS: Balsalazide and sulfasalazine were compared in a multicentre, double-blind, parallel group study over 12 weeks. Patients were stratified for disease severity and topical and/or oral steroids were co-administered where clinically necessary. RESULTS: Fifty-seven patients were randomized: 28 to receive balsalazide and 29 to receive sulfasalazine. Significantly fewer patients withdrew from the balsalazide group due to adverse events (2/28 vs. 9/29, P=0.041). These data confirm that balsalazide is better tolerated than sulfasalazine. In patients able to tolerate the treatment, similar improvements were recorded in clinical, sigmoidoscopic and histological assessments in both treatment groups. CONCLUSIONS: This study confirms the better tolerability of balsalazide compared to sulfasalazine, and supports the use of balsalazide in ulcerative colitis of all grades of severity.

A double-blind comparison of balsalazide, 6.75 g, and sulfasalazine, 3 g, as sole therapy in the management of ulcerative colitis.

BACKGROUND: Sulfasalazine is accepted therapy for active ulcerative colitis, but side-effects and intolerance are common. Balsalazide is an azo-bonded pro-drug which also releases 5-aminosalicylic acid into the colon, but uses an inert carrier molecule. AIM: To compare the safety and efficacy of sul- fasalazine, 3 g, with balsalazide, 6.75 g, in the initial daily treatment of mild to moderate ulcerative colitis. METHODS: A randomized, multicentre, double-blind, parallel group study was performed, with a treatment duration of 8 weeks. Patients on previous maintenance treatment were excluded. The trial medication was the sole treatment for the colitis. Efficacy was assessed by patient diaries, symptom assessment, sigmoidoscopic appearance and histology. RESULTS: Fifty patients were recruited: 26 allocated to the balsalazide group and 24 to the sulfasalazine group. More patients withdrew due to adverse events in the sulfasalazine group (nine patients vs. one patient in the balsalazide group, P=0.004). Improvement occurred in both groups, with a tendency to a faster response with balsalazide. Of the patients taking balsalazide, 61% achieved clinical and sigmoidoscopic remission. CONCLUSIONS: Balsalazide, 6.75 g, is effective as the sole treatment for patients with mild to moderately active ulcerative colitis, with significantly fewer withdrawals due to side-effects than in a similar group of patients taking sulfasalazine, 3 g.

Balsalazide in the maintenance treatment of patients with ulcerative colitis, a double-blind comparison with sulphasalazine.

Balsalazide (BSZ) is a pro-drug which releases 5-aminosalicylic acid (5ASA) and 4-aminobenzoyl-beta-alanine (an inert carrier) in the colon of various species including man. BSZ was compared with sulphasalazine (SASP) (both 1 g b.d. orally) in the maintenance of remission in patients with ulcerative colitis (UC). Seventy-nine patients (53 male, 26 female), mean age 49 years (range 19-79 years), with UC were randomly allocated to either treatment (41 BSZ, 38 SASP) for 6 months. The groups were similar in respect of age, sex, duration and extent of disease. Seven patients defaulted (3 BSZ, 4 SASP) leaving 38 on BSZ and 34 on SASP. Two male patients, both receiving SASP, were withdrawn because of severe side-effects. One of these patients, with an exfoliative rash, was maintained satisfactorily on open BSZ. Remission rates at 6 months (51% BSZ, 63% SASP) were not significantly different (life-table analysis P less than 0.1). Twelve patients (15%) reported troublesome side-effects (2 BSZ 5%, 10 SASP 26%, P = 0.017 Fisher Exact Test). Mean haemoglobin concentrations, similar on entry, increased after 6 months with BSZ (0.2 g/dl) but decreased with SASP (0.5 g/dl) (P less than 0.0002). BSZ was not significantly different from SASP in maintaining remission in patients with UC but had fewer side-effects.

Carbenoxolone interactions in man--preliminary report.

Carbenoxolone is a potent ulcer-healing drug which is extensively bound to plasma proteins and therefore has the potential for displacement interaction. However, carbenoxolone has been shown to be bound to human serum albumin in vitro at a different class of binding site to many other drugs and does not potentiate the pharmacological activity of warfarin, tolbutamide, chlorpropamide or phenytoin in the rat. In the present study four volunteers each received a single 100 mg dose of Biogastrone and the plasma half-life of carbenoxolone was determined. The procedure was repeated with a concurrent dose of either warfarin 10 mg, tolbutamide 500 mg, chlorpropamide 250 mg or phenytoin 100 mg. Chlorpropamide appeared to delay the absorption of carbenoxolone but no effects were observed with the other drugs. The study with concomitant chlorpropamide treatment was repeated with 6 gastric ulcer patients on an established Biogastrone regimen. In these patients the delayed absorption of carbenoxolone was confirmed although no changes in the glucose-lowering activity of chlorpropamide were evident. Further investigations into this findings are in progress.

The ferret, Mustela putorius furo, as a new species in toxicology.

Comprehensive studies on the haematology, urine chemistry, serum chemistry and gross pathology have been carried out and reported. Seasonal weight changes were confirmed and were initiated, together with sexual activity, when ferrets were brought in to a constant temperature (18-19 degrees C) and 14-hour daylight cycle during the winter months. Levels of haemoglobin, erythrocytes and haematocrit were higher than in most common laboratory species. Similarly, serum electrolytes and glucose levels were higher than in Wistar rats or beagle dogs. Ovaries, uteri and testes displayed a seasonal maturation and atrophy. All other parameters were similar to common laboratory species.