Properties of 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate--an intermediate in the non-mevalonate isoprenoid biosynthesis

Extraction and purification from the biomass of Corynebacterium ammoniagenes of 2-C-methyl-D-erhythritol 2,4-cyclopyrophosphate (MEC) was associated with its spontaneous transformation into a number of derivatives (which was due to pyrophosphate bond lability and the formation of complexes with metals). These derivatives included 1,2-cyclophospho-4-phosphate, 2,4-diphosphate, 2,3-cyclophosphate, 1,4-diphosphate, and 3,5-diphosphate (identified by 1H, 31P, and 13C NMR spectroscopy) and accounted for about 10% MEC. When added to a solution of DNA in the presence of the Fenton reagent, MEC prevented DNA decomposition. In addition, MEC slowed down the interaction of the reagent with tempol radicals, which indicates that complexation of ferrous ions by MEC attenuates their ability to catalyze the formation of hydroxyl radicals from hydrogen peroxide. In the presence of 0.23 mM MEC, the rate of respiration of rat liver mitochondria increased 1.8 times. At 0.1-1.0 mM, MEC activated in vitro proliferation of human Vgamma9 T-cells. It is suggested that MEC acts as an endogenous stabilizing agent for bacterial cells subjected to oxidative stress and as an immunomodulator for eukaryotic hosts.     

Effect of polyamines on synthesis and degradation of guanosine 5′-diphosphate 3′-diphosphate

The effect of polyamines on the in vitro and in vivo synthesis and degradation on guanosine 5′-diphosphate 3′-diphosphate (ppGpp) has been studied in Escherichia coli. The presence of 2 mM spermidine lowered the optimal Mg²⁺ concentration for ppGpp formation from 17 mM to 11 mM. The formation of ppGpp in the presence of 2 mM spermidine and 11 mM Mg²⁺ was about 15% greater than that in the presence of 17 mM Mg²⁺. At a concentration of less than 11 mM Mg²⁺, spermidine was found to stimulate ppGpp formation greatly. Putrescine did not cause any effect. When a polyamine-requiring mutant of E. coli (EWH319) was starved for an amino acid by the addition of valine, spermidine stimulated ppGpp formation. the degradation of ppGpp was not influenced significantly by polyamines.     

Purification,some properties and quaternary structure of thed-ribulose 1,5-diphosphate carboxylase ofAlcaligenes eutrophus

d-Ribulose 1,5-diphosphate carboxylase has been purified from autotrophically grown cells of the facultative chemolithotrophic hydrogen bacteriumAlcaligenes eutrophus. The enzyme was homogeneous by the criteria of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 505000 determined by gel filtration and sucrose density gradient centrifugation, and a sedimentation coefficient of 18.2 S was obtained. It was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis that the enzyme consists of two types of subunits of molecular weight 52000 and 13000.Electron microscopy on the intact and the partially dissociated enzyme lead to the construction of a model for the quaternary structure of the enzyme which is composed of 8 large and 8 small subunits. The most probable symmetry of the enzyme molecule is 4:2:2.Michaelis constant (K _m ) values for ribulose 1,5-diphosphate, Mg^2-, and CO₂ were 0.59 mM, 0.33 mM, and 0.066 mM measured under air. Oxygen was a competitive inhibitor with respect to CO₂ suggesting that the enzyme also exhibits an oxygenase activity. The oxygenolytic cleavage of ribulose 1,5-diphosphate was shown and a 1:1 stoichiometry between oxygen consumption and 3-phosphoglycerate formation observed.Abbreviations DTE dithioerythritol - EDTA ethylenediamine tetraacetate - RuDP d-ribulose 1,5-diphosphate     

DNA methylation by dimethyl sulfoxide and methionine sulfoxide triggered by hydroxyl radical and implications for epigenetic modifications

In this Letter, we demonstrate the formation of m⁵dC from dC or in DNA by dimethylsulfoxide (DMSO) and methionine sulfoxide (MetO), under physiological conditions in the presence of the Fenton reagent in vitro. DMSO reportedly affects the cellular epigenetic profile, and enhances the metastatic potential of cultured epithelial cells. The methionine sulfoxide reductase (Msr) gene was suggested to be a metastatis suppressor gene, and the accumulation of MetO in proteins may induce metastatic cancer. Our findings are compatible with these biological data and support the hypothesis that chemical cytosine methylation via methyl radicals is one of the mechanisms of DNA hypermethylation during carcinogenesis. In addition to m⁵dC, the formation of 8-methyldeoxyguanosine (m⁸dG) was also detected in DNA under the same reaction conditions. The m⁸dG level in human DNA may be a useful indicator of DNA methylation by radical mechanisms.     

Modification of Human Erythrocyte Pyruvate Kinase by an Active Site-directed Reagent: Bromopyruvate

Human erythrocyte pyruvate kinase was modified with bromopyruvate and the kinetic behavior of the modified enzyme was investigated. When the enzyme was modified with bromopyruvate in the absence of adenosine-5′s-diphosphate, phospho-enolpyruvate or fructose-1,6-diphosphate the inactivation followed a pseudo first-order kinetics. The inactivation rate constant, k_s, was 1.84 × 0.15 min^?1. K_d of the bromopyruvate-enzyme complex was 0.14 × 0.03 mM.

The presence of adenosine-5′-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate in the modification medium or the presence of fructose-1,6-diphosphate in the assay medium resulted in deviation of the inactivation kinetics from pseudo first-order. Phosphoenolpyruvate was better than adenosine-5′-diphosphate for protection against bromopyruvate modification whereas fructose-1,6-diphosphate was ineffective. The modified enzyme showed negative cooperativity in the presence of fructose-1,6-diphosphate whereas in the absence of it no activity was detected.     

Evidence for a uridine-5′-diphosphate-glucose-protectedp-chloromercuribenzene sulfonic acid-binding site in sugarcane vacuoles

The uptake of uridine-5-diphosphate (UDP) glucose into vacuoles isolated fromSaccharum sp. cells was fully inhibited by pretreatment with 50 Mp-chloromercuribenzenesulfonic acid (PCMBS) and was not affected by N-ethylmaleimide up to a concentration of 5 mM. The addition of 10 mM UDP-glucose during the pretreatment partially protected the uptake mechanism from PCMBS inhibition, while the presence of adenosine-5-diphosphate (ADP) glucose or of various hexose-phosphates had no protective effect. Parallel experiments on the binding of [²⁰³Hg]PCMBS to the vacuoles showed that UDP-glucose and UDP added at 10 mM concentrations caused a 40% decrease in the binding of PCMBS while ADP-glucose did not inhibit the binding. The results indicate the presence in a previously proposed group translocator of at least one site that can bind UDP-glucose. This site, which is blocked by PCMBS, interacts with the nucleotide moiety of UDP-glucose.Abbreviations ADP-glucose adenosine-5-diphosphate glucose - PCMB p-chloromercuribenzoic acid - PCMBS p-chloromercuribenzenesulfonic acid - UDP uridine-5-diphosphate - UDP-glucose uridine-5-diphosphate glucose     

Influence of auxins on organogenesis and ginsenoside production in Panax ginseng calluses

Panax ginseng calluses were cultured for 5 weeks on solid MS medium supplemented with kinetin 0.46 mM (0.1 mg l^–1) and 2 mg l^–1 of 2,4-D (9.05 mM), IBA (9.98 mM) or NAA (10.74 mM). In the conditions studied, 2,4-D inhibited the organogenic capacity of the calluses, whereas IBA or NAA increased this capacity. IBA induced the formation of a high number of buds and roots, but the roots were thin and necrotized. Calluses grown with NAA produced fewer buds and roots than those grown in IBA medium, but the roots were thick and showed good growth. The highest ginsenoside content was found in root forming calluses grown in the presence of NAA.In calluses forming roots or buds, 2,4-D, NAA and especially IBA increased the Rb group of ginsenosides rather than that of the Rg group.     

Purification and Characterisation of the Enantiospecific Dioxygenases from Delftia acidovorans MC1 Initiating the Degradation of Phenoxypropionate and Phenoxyacetate Herbicides

After cultivation on (R,S)‐2‐(2,4‐dichlorophenoxy)propionate, two α‐ketoglutarate‐dependent dioxygenases were isolated and purified from Delftia acidovorans MC1, catalysing the cleavage of the ether bond of various phenoxyalkanoate herbicides. One of these enzymes showed high specificity for the cleavage of the R‐enantiomer of substituted phenoxypropionate derivatives: the K_m values were 55 μM and 30 μM, the k_cat values 55 min^–1 and 34 min^–1 with (R)‐2‐(2,4‐dichlorophenoxy)propionate [(R)‐2,4‐DP] and (R)‐2‐(4‐chloro‐2‐methylphenoxy)propionate, respectively. The other enzyme predominantly utilised the S‐enantiomers with K_m values of 49 μM and 22 μM, and k_cat values of 50 min^–1 and 46 min^–1 with (S)‐2‐(2,4‐dichlorophenoxy)propionate [(S)‐2,4‐DP] and (S)‐2‐(4‐chloro‐2‐methylphenoxy)propionate, respectively. In addition, it cleaved phenoxyacetate herbicides (i.e. 2,4‐dichlorophenoxyacetate: K_m = 123 μM, k_cat = 36 min^–1) with significant activity. As the second substrate, only α‐ketoglutarate served as an oxygen acceptor for both enzymes. The enzymes were characterised by excess substrate inhibition kinetics with apparent K_i values of 3 mM with (R)‐2,4‐DP and 1.5 mM with (S)‐2,4‐DP. The reaction was strictly dependent on the presence of Fe²⁺ and ascorbate; other divalent cations showed inhibitory effects to different extents. Activity was completely extinguished within 2 min in the presence of 100 μM diethylpyrocarbonate (DEPC).     

Esr Spectra of Radicals of Single-Stranded and Double-Stranded DNA in Aqueous Solution. Implications for Oh-Induced Strand Breakage

In situ photolysis at 20^oC (argon plasma light source, $, $ 200 mm) of oxygen-free solutions containing 2mM H₂0₂ and heat-denatured, single-stranded (sS)DNA from calf-thymus resulted in the ESR spectra of the 6-hydroxy-5,6-dihydro-thymin-5-yl {1} and 5-methyleneuracil {3} radicals linked to the sugar-phosphate backbone. They were generated by reaction of OH radicals with DNA. By comparison of the decay characteristics of the ESR signals with rate constants from pulse-conductivity measurements [E. Bothe, G.A. Qureshi and D. Schulte-Frohlinde, Z. Naturforsch. 38c 1030, (1983)] the thymine-derived radicals {1} and {3} can be excluded as precursors of the fast, dominating component of strand breakage of ssDNA. In the absence of H₂0₂ from native, doubie-stranded (ds)DNA an ESR signal was obtained (singlet, g ～ 2.004, $1/2 ～ 0.8 mT) which was assigned to the deprotonated guanine radical cation, {G'(-H)} of a DNA subunit. It is assumed that by the UV irradiation the guanine radical cation, {G⁺}, is generated, either by monophotonic photoionisation or by electron transfer to pyrimidine bases. By rapid transfer of the bridging proton from {G⁺} to the hydrogen bonded cytosine {G'(-H)} is formed. When photolysis of dsDNA was carried out in the presence of H₂0₂, reaction of photolytically generated OH resulted in peroxyl radicals and purine radicals. The oxygen for formation of the peroxyl radicals is probably produced by reaction of {G' (-H)} with H₂0₂. Photolysis of N₂0-saturated solutions containing dsDNA or ssDNA provided another possibility of generation of OH radicals. Under those conditions the OH-induced radicals {1} and {3} were obtained not only from ssDNA but also from dsDNA.     

Oxidative Metabolism of 4-Aminobutyrate by Rat Brain Mitochondria: Inhibition by Branched-chain Fatty Acid

Abstract: The oxidation of 4-aminobutyric acid (GABA) by nonsynaptosomal mitochondria isolated from rat forebrain and the inhibition of this metabolism by the branched-chain fatty acids 2-methyl-2-ethyl caproate (MEC) and 2, 2-dimethyl valerate (DMV) were studied. The rate of GABA oxidation, as measured by O₂ uptake, was determined in medium containing either 5 or 100 mM-[K⁺]. The apparent K_m for GABA was 1.16 ± 0.19 mM and the V_max in state 3 was 23.8 ± 5.5 ng-atoms O₂. min^?1. mg protein^?1 in 5 mM-[K⁺]. In a medium with 100 mM-[K⁺] the apparent K_m was 1.11 ± 0.17 mM and V_max was 47.4 ± 5.7 ng-atoms O₂. min^?1. mg protein^?1. The K_m for MEC was determined to be 0.58 ± 0.24 or 0.32 ± 0.08 mM, in 5 or 100 mM-[K⁺], respectively. For DMV, the K_i was 0.28 ± 0.05 or 0.34 ± 0.06 mM, in 5 or 100 mM-[K⁺] medium, respectively. The O₂ uptake of the mitochondria in the presence of GABA was coupled to the formation of glutamate and aspartate; the ratio of oxygen uptake to the rate of amino acid formation was close to the theoretical value of 3. Neither the [K²] nor any of the above inhibitors had any effect on this ratio. The metabolism of exogenous succinic semialdehyde (SSA) by these same mitochondria was also examined. The V_max for utilization of oxygen in the presence of SSA was much greater than that found with exogenously added GABA, indicating that the capacity for GABA oxidation by these mitochondria is not limited by SSA dehydrogenase. In addition, the branched-chain fatty acids did not inhibit the metabolism of exogenously added SSA. Thus, the inhibitors examined apparently act by competitively inhibiting the GABA transaminase system of the mitochondria.     

A thiol-inducible and quick-response DNA cross-linking agent

Three new 2,4-dinitrobenzenesulfonyl derivatives 1–3 were successfully prepared for the first time using a simple process. They were efficiently triggered by thiols (glutathione and l-cysteine) to release the corresponding phenol derivatives (4–6) within 5?min. The quick response of 1–3 toward thiols was determined by ¹H NMR and HPLC. Moreover, our results indicated that 1 could induce DNA cross-linking in the presence of glutathione, probably due to the quinone methide formation of phenol intermediate 4 followed by departure of 2,4-dinitrobenzenesulfonyl group.     

Pyruvate Kinase of Streptococcus lactis

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V_max values and decreased the K_m values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The K_m values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.     

Induction of DNA synthesis by 2,4-dichlorophenoxyacetic acid during callus induction in Jerusalem artichoke tuber tissues

During the induction of DNA synthesis in Jerusalem artichoke (Helianthus tuberosus L.) tuber by 2,4-D, the 2-¹⁴C-2, 4-D from the agar medium rapidly incorporated into the ethanol soluble and insoluble fractions. Although the 2,4-D level in the ethanol soluble fraction decreased on transplantation of the tissue from the 2-¹⁴C-2,4-D medium to medium without the auxin, its level in the buffer-soluble and -insoluble macromolecular fractions increased. The purified, buffer-insoluble macromolecules were chromatin. The 2,4-D binding to chromatin particularly increased during DNA synthesis. The histone contents of chromatin decreased as DNA synthesis progressed. The polyacrylamide gel electrophoretic patterns of the histones showed a decrease in the moderately lysine-rich histone fraction as compared to other fractions. Thus, the decrease in the histone level caused by 2,4-D and the presence of the 2,4-D moderately lysine-rich histone complex may be closely related to the induction of DNA synthesis by 2,4-D in cells.     

Establishment of callus and cell suspension cultures from Gypsophila paniculata leaf segments and study of the attachment of host cells by Erwinia herbicola pv. gypsophilae

Callus and cell suspension cultures were initiated from leaf segments of G. paniculata. Fresh and dry weights measurements of callus showed that callus growth was optimal on MS medium supplemented with 1.0 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg l^–1 benzyladenin (BA). Calli cultured on this medium, showed a two-fold increase in fresh weight by the fourth week of incubation. The initiated hard green callus was repeatedly subcultured on MS medium containing increasing concentrations of 2,4-D in order to increase its friability. The friable callus was then used for establishment of a cell suspension culture. Maximum growth of the suspension culture was on medium supplemented with 1.0 mg l^–1 2,4-D and 0.2 mg l^–1 BA.The suspension culture was used for studying plant host attachment in both electron and light microscopy. Upon infection with E. herbicola, plant cells showed aggregate formation within 24 h of infection. In the presence of the pathogenic Ehg,the number of aggregates formed was 342 aggregates ml^–1, in the presence of the non-pathogenic Ehg154 aggregates ml^–1 and in the control 115 aggregates ml^–1. These results show that the pathogenic strain causes formation of cell aggregates 5.8 times greater than the non-pathogenic one. Based on these results, it can be hypothesized that bacterial cells of the pathogenic strains bind to the plant cells and may form a bridge for attachment of plant cells to one another. Observations by electron microscope show that bacterial cells do attach to plant cells and that this attachment might be via formation of a bridge between the bacteria and the plant cell.     

Production of UDP-N-acetylglucosamine by coupling metabolically engineered bacteria

A production system of UDP-N-acetylglucosamine (UDP-GlcNAc) was established by using recombinant Escherichia coli and Corynebacterium ammoniagenes in combination. E. coli overexpressed the UDP-GlcNAc biosynthetic genes, glmM, glmU, glk, ppa, ack, and pta, whereas C. ammoniagenes contributed to the formation of UTP from orotic acid. Glucose 1,6-diphosphate (Glc-1,6-P₂), which was required for the activity of phosphoglucosamine mutase involved in UDP-GlcNAc biosynthesis, was supplied by phosphoglucomutase and phosphofructokinase. Starting with orotic acid (65 mM) and glucosamine (400 mM), UDP-GlcNAc accumulated at 11.4 mM (7.4 g l^–1) after 8 h.     

The effect of SO 3 -- on the activity of ribulose-1,5-diphosphate carboxylase in isolated spinach chloroplasts

Summary SO ₃ ^-- inhibits the activity of ribulose-1,5-diphosphate carboxylase in isolated spinach chloroplasts. It shows a non-competitive inhibition pattern with respect to ribulose-1,5-diphosphate and Mg⁺⁺ but a competitive one with respect to HCO ₃ ^- . The K _i -values are 14 mM SO ₃ ^-- and 9.5 mM SO ₃ ^- respectively for the non-competitive inhibition but only 3.0 mM SO ₃ ^-- in the case of competitive inhibition with HCO ₃ ^-- as a substrate. Thus it is concluded that the competitive inhibition type will predominate at low SO ₃ ^-- and low internal CO₂ concentrations.The abbreviations used RuDph ribulose-1,5-diphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetate     

The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

Facile Synthesis of N-(1-Alkenyl) Derivatives of 2,4-Pyrimidinediones

Abstract

N-(1-alkenyl) derivatives of 2,4-pyrimidinediones (6–9) were prepared in a one pot synthesis from aldehydes and the nucleobases using trimethylsilyl trifluoromethanesulfonate (TfOTMS) as coupling reagent. Presilylation of the above nucleobases, and N ⁶-benzoyladenine, with excess N,O-bis(trimethylsilyl)acetamide (BSA) followed by addition of one mol eq. TfOTMS yielded the N-(1-trimethylsilyloxyalkyl) derivatives 1–5.     

Induction of astaxanthin formation by reactive oxygen species in mixotrophic culture of Chlorococcum sp.

The induction of astaxanthin formation by reactive oxygen species in mixotrophic culture of Chlorococcum sp. was investigated. H₂O₂ (0.1 mM) enhanced the total astaxanthin formation from 5.8 to 6.5 mg g^–1 cell dry wt. Fe²⁺ (0.5 mM) added to the medium with H₂O₂ (0.1 mM) further promoted astaxanthin formation to 7.1 mg g^–1 cell dry wt. Similarly, Fe²⁺ (0.5 mM) together with methyl viologen (0.01 mM) promoted astaxanthin formation to 6.3 mg g^–1 cell dry wt. In contrast, an addition of KI (1 mM), a specific scavenger for hydroxyl radicals (OH), together with H₂O₂ (0.1 mM) and Fe²⁺ (0.5 mM), to the medium decreased astaxanthin formation to 1.8 mg g^–1 cell dry wt. KI (1 mM) also inhibited the enhancement of carotenogenesis by superoxide anion radicals (O₂ ^–), with a decrease of astaxanthin formation to 1.7 mg g^–1 cell dry wt. This suggested that O₂ ^– might be transformed to OH before promoting carotenogenesis in Chlorococcum sp.     

G2 / M checkpoint genes of Saccharomyces cerevisiae : further evidence for roles in DNA replication and / or repair

We have cloned, sequenced and disrupted the checkpoint genes RAD17, RAD24 and MEC3 of Saccharomyces cerevisiae. Mec3p shows no strong similarity to other proteins currently in the database. Rad17p is similar to Rec1 from Ustilago maydis, a 3′ to 5′ DNA exonuclease/checkpoint protein, and the checkpoint protein Rad1p from Schizosaccharomyces pombe (as we previously reported). Rad24p shows sequence similarity to replication factor C (RFC) subunits, and the S. pombe Rad17p checkpoint protein, suggesting it has a role in DNA replication and/or repair. This hypothesis is supported by our genetic experiments which show that overexpression of RAD24 strongly reduces the growth rate of yeast strains that are defective in the DNA replication/repair proteins Rfc1p (cdc44), DNA polα (cdc17) and DNA polδ (cdc2) but has much weaker effects on cdc6, cdc9, cdc15 and CDC ⁺ strains. The idea that RAD24 overexpression induces DNA damage, perhaps by interfering with replication/repair complexes, is further supported by our observation that RAD24 overexpression increases mitotic chromosome recombination in CDC ⁺ strains. Although RAD17, RAD24 and MEC3 are not required for cell cycle arrest when S phase is inhibited by hydroxyurea (HU), they do contribute to the viability of yeast cells grown in the presence of HU, possibly because they are required for the repair of HU-induced DNA damage. In addition, all three are required for the rapid death of cdc13 rad9 mutants. All our data are consistent with models in which RAD17, RAD24 and MEC3 are coordinately required for the activity of one or more DNA repair pathways that link DNA damage to cell cycle arrest. Received: 8 April 1997 / Accepted: 10 May 1997