Control of dinucleoside polyphosphates by the FHIT-homologous HNT2 gene, adenine biosynthesis and heat shock in Saccharomyces cerevisiae
https://doi.org/10.1186/1471-2199-3-7
© Rubio-Texeira et al; licensee BioMed Central Ltd. 2002
Received: 31 January 2002
Accepted: 20 May 2002
Published: 20 May 2002
Abstract
Background
The FHIT gene is lost early in the development of many tumors. Fhit possesses intrinsic ApppA hydrolase activity though ApppA cleavage is not required for tumor suppression. Because a mutant form of Fhit that is functional in tumor suppression and defective in catalysis binds ApppA well, it was hypothesized that Fhit-substrate complexes are the active, signaling form of Fhit. Which substrates are most important for Fhit signaling remain unknown.
Results
Here we demonstrate that dinucleoside polyphosphate levels increase 500-fold to hundreds of micromolar in strains devoid of the Saccharomyces cerevisiae homolog of Fhit, Hnt2. Accumulation of dinucleoside polyphosphates is reversed by re-expression of Hnt2 and is active site-dependent. Dinucleoside polyphosphate levels depend on an intact adenine biosynthetic pathway and time in liquid culture, and are induced by heat shock to greater than 0.1 millimolar even in Hnt2+ cells.
Conclusions
The data indicate that Hnt2 hydrolyzes both ApppN and AppppN in vivo and that, in heat-shocked, adenine prototrophic yeast strains, dinucleoside polyphosphates accumulate to levels in which they may saturate Hnt2.
Keywords
Background
The human FHIT gene, located at the chromosome 3 fragile site FRA3B, is inactivated early in the development of many tumors [1]. Murine Fhit is also located at a fragile site [2, 3] and mice heterozygous for disruption of Fhit, given low intragastric doses of the mutagen N-nitrosomethylbenzylamine, develop stomach and sebaceous tumors [4] that can be prevented by viral Fhit expression [5]. Fhit, a dimer of 147 amino acid subunits, is a member of the histidine triad (HIT) superfamily of nucleotide hydrolases and transferases [6, 7]. Members of the Hint branch of the HIT superfamily are found in all forms of life [8]. The S. cerevisiae Hint homolog, Hnt1, and rabbit Hint possess adenosine monophosphoramidase activity that functions in yeast to positively regulate function of Kin28, Ccl1 and Tfb3, which constitute the kinase component of general transcription factor TFIIH [9]. A new Hint related protein, Aprataxin, is mutated in individuals with ataxia with oculomotor apraxia [10, 11] and has a yeast homolog termed Hnt3 [9]. Members of the Fhit branch of the HIT superfamily have been found in fungi [12, 13], animals [2, 14, 15] and plants [7] and hydrolyze diadenosine tetraphosphate, diadenosine triphosphate and other 5'-5"'-dinucleoside polyphosphates. The middle histidine of the histidine triad (His96 in human Fhit), which is critical for hydrolysis of ApppA by Fhit [14, 16], is not necessary for tumor suppression [17, 18]. Nonetheless, wild-type and His96Asn forms of Fhit are saturated by ApppA in the low micromolar range and form stable complexes with non-hydrolyzable ApppA in which two ApppA analogs are bound per Fhit dimer and all phosphates cluster on one surface of the protein [16]. These observations suggested that Fhit-substrate complexes may be the active, signaling form of Fhit and that the function of the catalytically essential histidine may be to terminate the lifetime of signaling complexes [16].
Given that neither transcriptional nor post-transcriptional regulation has been reported for Fhit protein, the level of biological activity of Fhit may be controlled by levels of Fhit substrates, inhibitors, and proteins that interact with Fhit-nucleotide complexes. Fhit proteins from humans [19] and worms [15] bind ApppA and AppppA with Km values of 2 to 3 μM. Human Fhit [14] and the S. cerevisiae Fhit homolog [13], which was called Aph1 but is here termed Hnt2 under nomenclature aproved by the Saccharomyces Genome Database, cleave ApppA more readily while Aph1, the S. pombe homolog, cleaves AppppA more readily [20]. Consistent with the ApppA hydrolase activity of purified Fhit protein, most cancer cell lines that are Fhit negative at the protein level have higher levels of ApppA than cell lines that are Fhit positive [21]. Nonetheless, the actual concentrations of dinucleoside polyphosphates were submicromolar in every cell culture sample [21] and thus, under the reported culture conditions, the measured dinucleoside polyphosphates would not be expected to occupy the Fhit active site substantially [19]. Dinucleoside polyphosphate levels were measured in adenine-requiring S. cerevisiae strains before or after disruption of the Fhit-homologous HNT2 gene [13] and in adenine-requiring S. pombe strains as a function of disruption and overexpression of the Fhit-homologous aph1 gene [22]. Recently, it was observed that diadenosine polyphosphates undergo a divalent cation-dependent conformational change that might mediate their biosynthesis, catabolism or signaling properties [23].
Here we discover a requirement of adenine biosynthesis for high-level dinucleoside polyphosphate accumulation in the absence of the Fhit homolog in S. cerevisiae. By constructing active site mutants of Hnt2 that were expressed in yeast, we demonstrate that ApppN and, to a lesser degree, AppppN levels are controlled by the Hnt2 active site. An added benefit of these constructions is the availability of yeast strains that possess high levels of dinucleoside polyphosphates and at the same time express a mutant Fhit-homologous protein, because these are conditions which have been postulated to constitute the signaling form of Fhit [16]. Finally, using controlled genotypes we revisited conditions that lead to increased accumulation of dinucleoside polyphosphates [24–33]. Recognizing that hnt2 deletion is a pathological condition, we were particular interested in identifying conditions that lead to accumulation of such compounds in cells that contain a functional HNT2 gene, rather than simply identifying conditions that produce diadenosine polyphosphate accumulation in the absence of Hnt2. While cells without a functional HNT2 gene accumulate dinucleoside polyphosphates in excess of 10 μM in a variety of nonstressed and stressed conditions, 46°C heat shock was the only condition that produced dinucleoside polyphosphate accumulation in excess of 10 μM in cells containing a functional HNT2 gene. These conditions did not render the cells conditionally null for Hnt2 because cells expressing HNT2 continued to limit dinucleoside polyphosphate accumulation during hours of heat shock, though at levels of ~0.1 mM.
Recently, discovery that the Hint-homologous HNT1 gene is required for high temperature growth on galactose and observations that alleles of cak1, kin28, ccl1 and tfb3 are hypersensitive to loss of Hnt1 enzyme activity provided evidence that Hnt1 enzyme activity positively regulates Kin28 function, particularly on galactose media [9]. Though phenotypic consequences of hnt2 mutations have yet to be discovered, our observations suggest that synthetic lethal interactions with hnt2 mutations are likely to be found in adenine prototrophic strains undergoing heat shock.
Results and discussion
Disruption of HNT2 and tetrad analysis of dinucleoside polyphosphate levels
S. cerevisiae strains in this study
Name | Genotype | Background and source |
---|---|---|
SEY6210 | MATα his3Δ200 leu2-3,112 lys 2-81 suc2-Δ9 trp1-Δ901 ura3-52 | S288C, [48] |
BY4717 | MA T a ade2Δ::hisG | S288C, [47] |
BY4727 | MATα his3Δ200 leu2Δ0 lys2Δ0 met15Δ0 trp1Δ63 ura3Δ0 | S288C, [47] |
BY16 | MATα his3Δ200 leu2Δ0 lys2Δ0 met15Δ0 trp1Δ63 ura3Δ0 hnt2Δ::kanMX2 | BY4727, this work |
BY71 | MAT a/MATα ADE2/ade2Δ::hisG HIS3/ his3Δ200 LEU2/leu2Δ0 LYS2/lys2Δ0 MET/met15Δ0 TRP1/trp1Δ63 URA3/ura3Δ0 HNT2/hnt2Δ::kanMX2 | BY4717 × BY16, this work |
BY71-1a | MATα his3Δ200 leu2Δ0 lys2Δ0 hnt2Δ::kanMX2 | BY71 segregant, this work |
BY71-1b | MATα ade2Δ::hisG his3Δ200 lys2Δ0 met15Δ0 trp1Δ63 ura3Δ0 hnt2Δ::kanMX2 | BY71 segregant, this work |
BY71-1c | MAT a ade2Δ::hisG leu2Δ0 met15Δ0 ura3Δ0 | BY71 segregant, this work |
BY71-1d | MA T a trp1Δ63 | BY71 segregant, this work |
BY71-2a | MATα ade2Δ::hisG leu2Δ0 met15Δ0 hnt2Δ::kanMX2 | BY71 segregant, this work |
BY71-2b | MATα his3Δ200 leu2Δ0 lys2Δ0 met15D0 ura3Δ0 hnt2Δ::kanMX2 | BY71 segregant, this work |
BY71-2c | MA T a ade2Δ::hisG his3Δ200 lys2Δ0 trp1Δ63 ura3Δ0 | BY71 segregant, this work |
BY71-2d | MA T a trp1Δ63 | BY71 segregant, this work |
BY71-4a | MAT a lys2Δ0 ura3Δ0 hnt2Δ::kanMX2 | BY71 segregant, this work |
BY71-4b | MATα his3Δ200 met15Δ0 ura3Δ0 | BY71 segregant, this work |
BY71-4c | MA T a ade2Δ::hisG his3Δ200 leu2Δ0 met15Δ0 trp1Δ63 | BY71 segregant, this work |
BY71-4d | MATα ade2Δ::hisG leu2Δ0 lys2Δ0 trp1Δ63 hnt2Δ::kanMX2 | BY71 segregant, this work |
BY71-6c | MAT a his3Δ200 leu2Δ0 met15Δ0 hnt2Δ::kanMX2 | BY71 segregant, this work |
BY71-16d | MAT a his3Δ200 leu2Δ0 met15Δ0 | BY71 segregant, this work |
Disruption of hnt2. Strain BY16 was crossed with strain BY4717 to generate diploid strain BY71, which was dissected to generate haploid progeny. Markers were scored for mating type, auxotrophic requirements and G418-resistance. The two small segregants per tetrad are ade2Δ mutants. Here, four segregants that had been scored for G418-resistance were scored for size of the HNT2 locus by PCR using diagnostic primers 4722 and 4726. G418-sensitive progeny, BY71-3a and BY71-3c produced a product of 1200 bp while G418-resistant progeny BY71-3b and BY71-3d produced a product of 1976 bp, demonstrating physical linkage of kanMX2 to hnt2 disruption.
Intracellular concentration (μM) of dinucleoside polyphosphates in segregants from three complete BY71 tetrads
Culture Time | 24 hr | 48 hr | 72 hr | ||||
---|---|---|---|---|---|---|---|
Nucleotide | ApppN | AppppN | ApppN | AppppN | ApppN | AppppN | |
Segregant | Relevant Genotype | ||||||
1a | 5.96 | 0.27 | 112 | 0.26 | 251 | 0.19 | hnt2Δ ADE2 |
1b | 14.2 | 0.11 | 42.7 | 0.12 | 101 | 0.16 | hnt2Δ ade2 |
1c | 1.44 | 0.16 | 2.86 | 0.09 | 2.50 | 0.09 | HNT2 ade2 |
1d | 0.26 | 0.04 | 1.07 | 0.38 | 0.60 | 0.16 | HNT2 ADE2 |
2a | 42.6 | 0.23 | 86.6 | 0.19 | 102 | 0.18 | hnt2Δ ade2 |
2b | 39.1 | 0.37 | 294 | 1.51 | 346 | 0.26 | hnt2Δ ADE2 |
2c | 0.29 | 0.06 | 0.40 | 0.05 | 0.36 | 0.05 | HNT2 ade2 |
2d | 2.59 | 0.17 | 0.86 | 0.21 | 0.57 | 0.07 | HNT2 ADE2 |
4a | 21.6 | 0.52 | 160 | 0.37 | 200 | 0.42 | hnt2Δ ADE2 |
4b | 0.51 | 0.09 | 0.24 | 0.002 | 0.09 | 0.001 | HNT2 ADE2 |
4c | 0.34 | 0.07 | 0.95 | 0.03 | 0.80 | 0.06 | HNT2 ade2 |
4d | 8.21 | 0.10 | 26.7 | 0.07 | 52.1 | 0.19 | hnt2Δ ade2 |
Earlier, hnt2 deletion was reported to increase AppppN levels only 2.5-fold but the study was performed in ade2 mutants [13]. Consistent with that report, the three hnt2 ade2 strains showed only a 2-fold higher AppppN level than the three HNT2 ade2 strains, when nucleotide levels were averaged across the three time points. In contrast, hnt2ΔADE2 strains achieved a 3.7-fold higher level of AppppN than HNT2 ADE2 strains. Larger increases in AppppN concentrations have been seen with disruption of Apa1 and Apa2, the diadenosine tetraphosphate phosphorylases in S. cerevisiae[34, 35], indicating that they have a more significant role in controlling AppppN levels than does Hnt2. In the case of disruption of the Fhit and Hnt2-homologous aph1 gene in S. pombe, which encodes an enzyme relatively specific for a AppppA [20], a 290-fold increase in AppppA concentration was observed [22]. Our data indicate that Hnt2 hydrolyzes ApppN and AppppN in vivo in budding yeast and that an intact adenine biosynthetic pathway is required for high-level synthesis and accumulation of adenylylated dinucleoside polyphosphates.
Hnt2 active site-dependence of dinucleoside polyphosphate accumulation
Plasmids used in this study
Name | Features | Background and source |
---|---|---|
pRS423 | YEp HIS3 | [39] |
pB05 | YEp HIS3 HNT2 | pRS423, this work |
pB32 | YEp HIS3 HNT2-His109Ala | pB05, this work |
pB86 | YEp HIS3 HNT2-His109Asp | pB05, this work |
pM1 | YEp HIS3 KRS1 | pRS423, this work |
Intracellular concentration (μM) of dinucleoside polyphosphates controlled by the Hnt2 active site
Culture time | 24 hr | 48 hr | |||
---|---|---|---|---|---|
Nucleotide | ApppN | AppppN | ApppN | AppppN | |
Plasmid in strain BY71-6c | Genotype | ||||
None | 20.70 | 0.16 | 129.00 | 2.16 | hnt2Δ |
pRS423 | 23.80 | 0.21 | 140.00 | 1.11 | hnt2Δ |
pB05 | 0.51 | 0.07 | 1.11 | 0.32 | HNT2 |
pB32 | 18.70 | 0.17 | 201.00 | 1.69 | HNT2-His109Ala |
pB86 | 13.40 | 0.16 | 80.40 | 1.00 | HNT2-His109Asp |
Dinucleoside polyphosphate levels may not be limited by the levels of lysyl tRNA synthetase
Intracellular concentration (μM) of dinucleoside polyphosphates as a function of culture time, HNT2 genotype, and presence of multicopy lysyl-tRNA synthetase gene
Nucleotide | ApppN | AppppN | ||||
---|---|---|---|---|---|---|
Culture Time | 24 hr | 48 hr | 72 hr | 24 hr | 48 hr | 72 hr |
Relevant Genotype | ||||||
HNT2 | 0.52 | 1.60 | 0.22 | 0.29 | 0.19 | 0.02 |
HNT2 YEpKRS1 | 1.28 | 1.29 | 0.20 | 0.34 | 0.22 | 0.01 |
hnt2Δ | 6.25 | 14.10 | 3.54 | 0.35 | 0.26 | 0.02 |
hnt2Δ YEpKRS1 | 5.83 | 15.50 | 4.20 | 0.40 | 0.30 | 0.01 |
Moderate overexpression of lysyl tRNA synthetase activity via multicopy plasmid pM1 Lysates from strain BY71-6c transformants with control plasmid pRS423 and multicopy KRS1 plasmid pM1 were assayed for incorporation of 3H lysine in the absence and presence of added yeast tRNA.
Heat shock is the most effective stress for elevation of dinucleoside polyphosphates
In the wild and in the laboratory, yeast are exposed to stresses such as hypo-osmotic or hyperosmotic conditions, toxic cations, heat shock and cell-cycle disruptive reagents. To test whether such conditions induce dinucleoside polyphosphates in hnt2- or Hnt2+ cells, we incubated ADE2 hnt2 and ADE2 HNT2 cells in water, 1 M sorbitol, 2 mM CdCl2, 46 °C heat shock, 10 mM caffeine, or in rich media for two hours and determined dinucleoside polyphosphate levels. Additionally, to test whether moderate overexpression of the lysyl tRNA synthetase gene affected accumulation, we compared control transformants to multicopy KRS1 transformants of the two strains. As shown in Table 6, the hnt2 samples had substantially higher ApppN levels than HNT2 samples under all conditions. Among the hnt2 samples, only heat shocked samples showed evidence of ApppN levels higher than the levels in nonstressed hnt2 cells. Similarly, among the HNT2 samples, the heat shocked samples showed increased ApppN levels compared with control-treated cells while CdCl2 and other treated samples showed no significant changes. KRS1 on a multicopy plasmid showed no significant alteration of ApppN levels in any sample. As with other experiments, AppppN levels were lower than ApppN levels in all cases. Heat shock was the best inducer of AppppN. Hypotonic, hypertonic and caffeine treated media produced no increase in AppppN (not shown).
To further investigate the kinetics of heat shock and cadmium-induction of ApppN and AppppN levels, we transformed hnt2ΔADE2 strain BY71-6c with multicopy plasmids containing no HNT2 gene, the wild-type HNT2 gene, or the HNT2-His109Ala or HNT2-His109Asp alleles of HNT2. Cultures were exposed to either 2 mM CdCl2 or 46°C heat shock and intracellular concentrations of ApppN and AppppN were determined at 30-minute timepoints.
Cadmium is a poor inducer of dinucleoside polyphosphates Calculated intracellular concentrations of ApppN and AppppN of transformants of strain BY71-6c as a function of time in 2 mM CdCl2.
Heat shock induces dinucleoside polyphosphates Calculated intracellular concentrations of ApppN and AppppN of transformants of strain BY71-6c as a function of time at 46°C.
Conclusions
It had been reported that Hnt2 controls ApppN levels in vivo, with a minor effect on AppppN [13]. Here we show that the ade2 mutation present in earlier experiments prevents accumulation of ApppN and AppppN and reduced the magnitude of the Hnt2 effect. In ADE2 strains examined herein, deletion of HNT2 increased levels of ApppN and AppppN by factors of approximately 200 and 4, respectively. Mutagenesis [14], X-ray crystallography [16, 40], and stereochemical analysis [41] indicate that His96 is the nucleophile that attacks the α-phosphate of Fhit substrates. Our analysis shows that the corresponding residue, His109 of Hnt2, is required for hydrolysis of ApppN and AppppN substrates in vivo.
Other than effects on the concentrations of intracellular nucleotides, neither deletion of HNT2 nor mutation of His109 of HNT2 had phenotypic consequences. In the case of the Hint-homologous HNT1 gene of S. cerevisiae, the phenotype of the single mutant was mild and the biological pathway, positive regulation of Kin28, the yeast homolog of Cdk7, was revealed by synthetic lethal interactions between hnt1 and hypomorphic alleles of cak1, kin28, ccl1 and tfb3[9]. Because backup systems to limit problems with hnt2 mutant cells apparently exist, synthetic lethal interactions may be critical to identify the Hnt2 biological pathway. Thus, if the major function of HNT2 is simply control of dinucleoside polyphosphates lest their accumulation inhibit other enzymes (i.e., McLennan's "foe" hypothesis [42]), then synthetic lethal phenotypes ought to be sought with hnt2 mutant strains that are wild-type for the adenine biosynthetic pathway, potentially under heat shock conditions. On the other hand, if the major function of Hnt2 depends on formation of an enzyme-substrate complex [16], then synthetic lethal interactions ought to be sought in heat shocked ADE2 HNT2-His109Ala strains. In fact, because ADE2 HNT2-His109Ala strains have high levels of dinucleoside polyphosphates and an Hnt2 polypeptide, such a strain may be sensitized to secondary mutations, whether dinucleoside polyphosphates are friend or foe [42].
Materials and methods
General molecular biology
Yeast media and procedures were as described [43, 44]. S. cerevisiae transformations were carried out by the lithium acetate method [45]. E. coli strain XL-1 Blue was used for bacterial cloning and plasmid amplification. Bacterial media and molecular biology techniques were as described [44].
Disruption of hnt2
Intracellular concentration (μM) of ApppN as a function of stress treatments, HNT2 genotype, and moderate overexpression of lysyl aminoacyl-tRNA synthetase
Relevant Genotype | -his | 1 M sorbitol | 10 mM caffeine | 2 mM CdCl2 | 46°C | H2O | YPD |
---|---|---|---|---|---|---|---|
HNT2 | 0.58 ± 0.36 | 1.19 ± 0.37 | 1.15 ± 0.12 | 1.12 ± 0.29 | 2.16 ± 0.10 | 0.54 ± 0.12 | 0.44 ± 0.09 |
HNT2 YEpKRS1 | 0.60 ± 0.51 | 0.84 ± 0.21 | 2.74 ± 1.37 | 1.14 ± 0.17 | 2.11 ± 0.25 | 0.49 ± 0.05 | 0.41 ± 0.13 |
hnt2Δ | 10.7 ± 7.51 | 6.65 ± 0.82 | 12.5 ± 3.91 | 4.92 ± 2.67 | 44.2 ± 7.51 | 5.92 ± 1.66 | 5.97 ± 1.38 |
hnt2Δ YEpKRS1 | 19.0 ± 8.21 | 6.36 ± 2.10 | 3.84 ± 1.05 | 11.6 ± 4.36 | 40.6 ± 5.62 | 8.82 ± 3.57 | 1.40 ± 0.52 |
Plasmid constructions
The HNT2 gene was amplified from genomic DNA of yeast strain SEY6210 [48] with primers PB1 (5'GCAGCGGATCCTTGGGAT) that spanned a Bam HI site upstream of the promoter and PB2 (5'GAGTCTCCTCGAGGAAAG) that spanned a Xho I site downstream of the terminator. The 1316 bp Bam HI-Xho I fragment containing HNT2 was ligated to Bam HI and Xho I-cleaved plasmid pRS423 [39] to generate plasmid pB05. Plasmids pB32 and pB86, carrying H109A and H109D alleles of HNT2, were constructed by site-directed mutagenesis [49] of plasmid pB05 using primers PB3 (5'ATAATGTGTGTAGCCAAGTGGGGT) and PB4 (5'TAATGTGTGTATCCAAGTGGGGTAC). The S. cerevisiae gene encoding lysyl tRNA synthetase (KRS1) was amplified as a 2.9 kbp genomic fragment from strain SEY6210 using primers MR20 (5'CGAGCTCGGTTGGA TGACTTTAAAATGACTAAGTTTGTAGTATCCTCTTTGC ATACTC) and MR21 (5'TCCCCCGGGGGAGCTCCTTTAGGGCTACCGAACATAAACAAATTTAGGTAA TGAGTTTTC). This product, cloned into the Sma I restriction site of plasmid pRS423, generated plasmid pM1 in which KRS1 is oriented anti to HIS3. Plasmids are summarized in Table 3.
Measurement of dinucleoside polyphosphate levels
Twelve haploid segregants, pregrown in liquid SDC medium, were inoculated into 250 ml of SDC at starting density of 104 cells per ml. At 24, 48 and 72 hours of growth, 50 ml of cells were harvested, cells counted microscopically, lysed, and levels of AppppN and ApppN were determined as described [13]. Intracellular concentrations of ApppN and AppppN were calculated using 7 × 10-14 l as the volume of a haploid cell [50]. Cultures of BY71-6c were transformed with plasmids pRS423, pB05, pB32 and pB86 and transformants were selected on SDC-his media. Intracellular concentrations of ApppN and AppppN were determined for transformants from cultures in SDC-his media as above. To determine whether a multicopy lysyl tRNA synthetase plasmid affected accumulation of dinucleoside polyphosphates, we transformed HNT2 ADE2 strain BY71-16d and hnt2ΔADE2 strain BY71-6c with control plasmid pRS423 and with plasmid pM1. Transformants were grown for 24, 48 and 72 hours in SDC-his media and intracellular dinucleoside polyphosphate concentrations were determined as above. To survey dinucleoside polyphosphate induction as a function of potential stress conditions, strain BY71-6c was transformed with either pRS423 or pM1 (effectively hnt2Δ and hnt2Δ YEpKRS1, respectively) and strain BY71-16d was transformed with the same plasmids (effectively HNT2Δ and HNT2Δ YEpKRS1, respectively). After 48 hours of culture, cells were pelleted and resuspended in either YPD media, water, SDC-his media, or the same media supplemented with 2 mM CdCl2, 10 mM caffeine or 1 M sorbitol. The SDC-his sample was incubated for 2 hours at 46°C while other samples were incubated for 2 hours at room temperature prior to extraction for determination of dinucleoside polyphosphate concentrations. To determine the time course of ApppN and AppppN levels as a function of stresses, we used strains BY71-16d and BY71-6c transformed with pRS423, pB05, pB32, or pB86. Transformants were cultured for 48 hours, treated with 2 mM CdCl2 or 46°C heat shock, and then harvested for nucleotide quantitation at 30 minute intervals. Experiments presented in Tables 2, 4, 5 and 6 were performed two, three, five and three times, respectively. Experiments presented in Figures 3 and 4 were performed four times each. Because diadenosine polyphosphate levels vary with time in culture, generating a higher or lower range of values in independently conducted experiments, we did not average values obtained in separate experiments. For the data presented in Table 6, triplicate cultures were prepared and the ApppN levels for identically treated samples are provided as averages ± standard deviations.
Lysyl tRNA synthetase activity assay
Strain BY71-6c was transformed with plasmids pRS423 and pM1 (multicopy KRS1) and cultures were grown as for measurement of dinucleoside polyphosphate levels. Lysates were prepared by glass bead disruption in 50 mM Tris Cl pH 7.5, 1 mM DTT, 40% glycerol. Incorporation of tritiated lysine into tRNA was measured by modification of the protocol of Hou [51]. Reactions contained 10 micrograms of total protein in 20 mM KCl, 10 mM MgCl2, 4 mM dithiothreitol, 2 mM ATP, 50 mM Na HEPES pH 7.5, 20 μM lysine (10 μCi 3H lysine), and were performed with or without 15 μg yeast tRNA (Sigma) in a total volume of 60 μl. At one, two, three, five and twelve minute time points, 10 μl aliquots were spotted on filter paper and placed in 5% wt/vol trichloroacetic acid, washed in 5% trichloroacetic acid twice, washed twice in 95% ethanol, rinsed in ether and scintillation-counted.
Authors' note
Tetrads shown in figure 1 were dissected and PCR analyzed to show the procedure followed to obtain strains. They do not correspond to tetrads mentioned in table 1.
Declarations
Acknowledgements
This work was supported by a grant from the National Cancer Institute (CA75954). We thank Dr. Ya-Ming Hou and her laboratory for guidance in measuring lysyl tRNA synthetase activity and an anonymous reviewer for helpful suggestions.
Authors’ Affiliations
References
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