Cyanide, Peroxide and Nitric Oxide Formation in Solutions of Hydroxyurea Causes Cellular Toxicity and May Contribute to Its Therapeutic Potency

Abstract

Hydroxyurea is a potent remedy against a variety of ailments and an efficient inhibitor of DNA synthesis, yet its pharmacology is unclear. Hydroxyurea acts in Escherichia coli by the same mechanism as it does in eukaryotes, via inhibition of ribonucleotide reductase. When examining a controversy about concentrations of hydroxyurea that prevent thymineless death in E. coli, we found instability in hydroxyurea solutions which avoided prior detection due to its peculiar nature. In contrast to freshly dissolved hydroxyurea, which did not affect respiration and was bacteriostatic, one-day-old hydroxyurea solutions inhibited respiration and were immediately bactericidal. Respiration was inhibited by two gasses, hydrogen cyanide (HCN) and nitric oxide (NO), whose appearance we detected in “aged” hydroxyurea stocks by GC-MS; however, neither gas was bactericidal. While determining the cause of toxicity, we found that hydroxyurea damages DNA directly. We also demonstrated accumulation of peroxides in hydroxyurea solutions by enzymatic assays, which explains the toxicity, as both NO and HCN are known to kill bacteria when combined with hydrogen peroxide. Remarkably, we found that bactericidal effects of NO + H2O2 and HCN + H2O2 mixtures were further synergistic. Accumulation of decomposition products in solutions of hydroxyurea may explain the broad therapeutic effects of this drug.

Disintegration of Nascent Replication Bubbles during Thymine Starvation Triggers RecA- and RecBCD-dependent Replication Origin Destruction

“…1B) depends on the replication fork progress in T-starved cells. Because DNA replication in T-starved cells was never measured directly via incorporation (see above for the reason), we developed a novel protocol to measure DNA synthesis in T-starved cells by employing [ 32 P]orthophosphate, lysing cells in agarose plugs, and separating DNA from other 32 P-labeled species by alkaline gel electrophoresis (26). Using continuous 32 P labeling, we found that during the first 30 min of thymine starvation the accumulation of 32 P label in chromosomal DNA in the absence of thymidine is only three times lower than in cells growing in the presence of thymidine, although this difference becomes 25-fold after a 4.5-h incubation (Fig.…”

“…The following strains were used: ⌬thyA, KKW58; Thy ϩ , KKW59; ⌬recA, KKW60; ⌬thyA ⌬recA, KJK61; ⌬thyA ⌬recBCD, KJK63; dut recBC(Ts), AK107; they are all published (10,26,27) and all, with the exception of AK107, carry deletion of the entire deoCABD operon. Alleles were moved between strains by P1 transduction (28,29).…”

“…Recently, we described a novel method of DNA labeling during T-starvation using [ 32 P]orthophosphoric acid (26). Here we employed this new DNA labeling technique, together with differential labeling for chromosomal fragmentation analysis with pulsed-field gels, as well as the marker frequency analysis with gene arrays, to show how the postulated single-strand gaps are converted into irreparable chromosomal lesions by toxic repair during T-starvation.…”

Replication Forks Stalled at Ultraviolet Lesions Are Rescued via RecA and RuvABC Protein-catalyzed Disintegration in Escherichia coli

“…Chromosomal Fragmentation-In our protocol, quantification of chromosomal fragments is facilitated by 32 P labeling of DNA; other 32 P-labeled species, such as RNA, polyphosphates, LPS, and phospholipids are removed either during plug preparation or during the electrophoretic run (42). The amount of 32 P label that we use is not enough to affect the viability of even the most DNA damage-sensitive mutants, like uvrA recA double mutants.…”

Static and Dynamic Factors Limit Chromosomal Replication Complexity inEscherichia coli, Avoiding Dangers of Runaway Overreplication

“…A defect in nucleotide reductase, the enzyme that supplies replication forks with DNA precursors, also increases the ori/ter ratio to 5 (Salguero et al 2011). A nucleotide reductase inhibitor, hydroxyurea (HU), reduces DNA replication rate in E. coli to 20% of the uninhibited rate (Kuong and Kuzminov 2009) and, within 4 hr, brings the ori/ter ratio to 5 (Kuong and Kuzminov 2012). Mutants in the rep have gene slow Figure 1 Increased replication complexity in cells with slow replication forks reveals the "natural" CRC limit.…”