Metabolic reprogramming driven by EZH2 inhibition depends on cell-matrix interactions.

EZH2 (Enhancer of Zeste Homolog 2), a subunit of Polycomb Repressive Complex 2 (PRC2), catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3), which represses expression of genes. It also has PRC2-independent functions, including transcriptional coactivation of oncogenes, and is frequently overexpressed in lung cancers. Clinically, EZH2 inhibition can be achieved with the FDA-approved drug EPZ-6438 (tazemetostat). To realize the full potential of EZH2 blockade, it is critical to understand how cell-cell/cell-matrix interactions present in 3D tissue and cell culture systems influences this blockade in terms of growth-related metabolic functions. Here, we show that EZH2 suppression reduced growth of human lung adenocarcinoma A549 cells in 2D cultures but stimulated growth in 3D cultures. To understand the metabolic underpinnings, we employed [13C6]-glucose stable isotope-resolved metabolomics to determine the effect of EZH2 suppression on metabolic networks in 2D versus 3D A549 cultures. The Krebs cycle, neoribogenesis, γ-aminobutyrate metabolism, and salvage synthesis of purine nucleotides were activated by EZH2 suppression in 3D spheroids but not in 2D cells, consistent with the growth effect. Using simultaneous 2H7-glucose + 13C5,15N2-Gln tracers and EPZ-6438 inhibition of H3 trimethylation, we delineated the effects on the Krebs cycle, γ-aminobutyrate metabolism, gluconeogenesis, and purine salvage to be PRC2-dependent. Furthermore, the growth/metabolic effects differed for mouse Matrigel versus self-produced A549 extracellular matrix. Thus, our findings highlight the importance of the presence and nature of extracellular matrix in studying the function of EZH2 and its inhibitors in cancer cells for modeling the in vivo outcomes.

A plastid nucleoside kinase is involved in inosine salvage and control of purine nucleotide biosynthesis.

In nucleotide metabolism, nucleoside kinases recycle nucleosides into nucleotides-a process called nucleoside salvage. Nucleoside kinases for adenosine, uridine, and cytidine have been characterized from many organisms, but kinases for inosine and guanosine salvage are not yet known in eukaryotes and only a few such enzymes have been described from bacteria. Here we identified Arabidopsis thaliana PLASTID NUCLEOSIDE KINASE 1 (PNK1), an enzyme highly conserved in plants and green algae belonging to the Phosphofructokinase B family. We demonstrate that PNK1 from A. thaliana is located in plastids and catalyzes the phosphorylation of inosine, 5-aminoimidazole-4-carboxamide-1-ß-d-ribose (AICA ribonucleoside), and uridine but not guanosine in vitro, and is involved in inosine salvage in vivo. PNK1 mutation leads to increased flux into purine nucleotide catabolism and, especially in the context of defective uridine degradation, to over-accumulation of uridine and UTP as well as growth depression. The data suggest that PNK1 is involved in feedback regulation of purine nucleotide biosynthesis and possibly also pyrimidine nucleotide biosynthesis. We additionally report that cold stress leads to accumulation of purine nucleotides, probably by inducing nucleotide biosynthesis, but that this adjustment of nucleotide homeostasis to environmental conditions is not controlled by PNK1.

Horizontal transfer of a pathway for coumarate catabolism unexpectedly inhibits purine nucleotide biosynthesis.

A microbe's ecological niche and biotechnological utility are determined by its specific set of co-evolved metabolic pathways. The acquisition of new pathways, through horizontal gene transfer or genetic engineering, can have unpredictable consequences. Here we show that two different pathways for coumarate catabolism failed to function when initially transferred into Escherichia coli. Using laboratory evolution, we elucidated the factors limiting activity of the newly acquired pathways and the modifications required to overcome these limitations. Both pathways required host mutations to enable effective growth with coumarate, but the necessary mutations differed. In one case, a pathway intermediate inhibited purine nucleotide biosynthesis, and this inhibition was relieved by single amino acid replacements in IMP dehydrogenase. A strain that natively contains this coumarate catabolism pathway, Acinetobacter baumannii, is resistant to inhibition by the relevant intermediate, suggesting that natural pathway transfers have faced and overcome similar challenges. Molecular dynamics simulation of the wild type and a representative single-residue mutant provide insight into the structural and dynamic changes that relieve inhibition. These results demonstrate how deleterious interactions can limit pathway transfer, that these interactions can be traced to specific molecular interactions between host and pathway, and how evolution or engineering can alleviate these limitations.

Epitranscriptomic Code and Its Alterations in Human Disease.

Innovations in epitranscriptomics have resulted in the identification of more than 160 RNA modifications to date. These developments, together with the recent discovery of writers, readers, and erasers of modifications occurring across a wide range of RNAs and tissue types, have led to a surge in integrative approaches for transcriptome-wide mapping of modifications and protein-RNA interaction profiles of epitranscriptome players. RNA modification maps and crosstalk between them have begun to elucidate the role of modifications as signaling switches, entertaining the notion of an epitranscriptomic code as a driver of the post-transcriptional fate of RNA. Emerging single-molecule sequencing technologies and development of antibodies specific to various RNA modifications could enable charting of transcript-specific epitranscriptomic marks across cell types and their alterations in disease.

Optimization of the purine operon and energy generation in Bacillus amyloliquefaciens for guanosine production.

OBJECTIVES: To deregulate the purine operon of the purine biosynthetic pathway and optimize energy generation of the respiratory chain to improve the yield of guanosine in Bacillus amyloliquefaciens XH7. RESULTS: The 5'-untranslated region of the purine operon, which contains the guanine-sensing riboswitch, was disrupted. The native promoter Pw in B. amyloliquefaciens XH7 was replaced by different strong promoters. Among the promoter replacement mutants, XH7purE::P41 gave the highest guanosine yield (16.3 g/l), with an increase of 23% compared with B. amyloliquefaciens XH7. The relative expression levels of the purine operon genes (purE, purF, and purD) in the XH7purE::P41 mutant were upregulated. The concentration of inosine monophosphate (IMP), the primary intermediate in the purine pathway, was also significantly increased in the XH7purE::P41 mutant. Combined modification of the low-coupling branched respiratory chains (cytochrome bd oxidase) improved guanosine production synergistically. The final guanosine yield in the XH7purE::P41△cyd mutant increased by 41% to 19 g/l compared with B. amyloliquefaciens XH7. CONCLUSION: The combined modification strategy used in this study is a novel approach to improve the production of guanosine in industrial bacterial strains.

Chronic alcohol administration affects purine nucleotide catabolism in vivo.

AIMS: To investigate the relationship between chronic alcohol administration and purine nucleotide metabolism in vivo. MAIN METHODS: Rat models of alcohol dependence and withdrawal were used. The concentrations of uric acid (UAC), urea nitrogen (UREA), creatinine (CREA), and beta-2-microglobulin (ß2-M) and creatinine clearance rate (CCR) in plasma were measured. The PLC method was used to detect the absolute content of purine nucleotides in different tissues. Enzymatic activities of adenosine deaminase (ADA), xanthine oxidase (XO), ribose 5-phosphate pyrophosphokinase (RPPPK), glutamine phosphoribosylpyrophosphate amidotransferase (GPRPPAT), hypoxanthine-guanine phosphate ribose transferase (HGPRT), and adenine phosphoribosyltransferase (APRT) in the tissues were analyzed. Real-time PCR was used to determine the relative level of ADA and XO. KEY FINDINGS: The renal function of rats with alcohol dependence was normal. Further, the content of purine nucleotides (GMP, AMP, GTP, and ATP) in tissues of the rats was decreased, which indicated that the increased uric acid should be derived from the decomposition of nucleotides in vivo. The activity of XO and ADA increased, and their mRNA expression was enhanced in the alcohol dependence group, but there was no significant difference in the activity of RPPPK and GPRPPAT in the liver, small intestine, and muscle; furthermore, no significant difference in the activity of HGPRT and APRT was observed in the brain. SIGNIFICANCE: These results indicate that chronic alcohol administration might enhance the catabolism of purine nucleotides in tissues by inducing gene expression of ADA and XO, leading to elevation of plasma uric acid levels.

Aspartate Rescues S-phase Arrest Caused by Suppression of Glutamine Utilization in KRas-driven Cancer Cells.

During G1-phase of the cell cycle, normal cells respond first to growth factors that indicate that it is appropriate to divide and then later in G1 to the presence of nutrients that indicate sufficient raw material to generate two daughter cells. Dividing cells rely on the "conditionally essential" amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates and as a nitrogen source for nucleotide biosynthesis. We previously reported that while non-transformed cells arrest in the latter portion of G1 upon Q deprivation, mutant KRas-driven cancer cells bypass the G1 checkpoint, and instead, arrest in S-phase. In this study, we report that the arrest of KRas-driven cancer cells in S-phase upon Q deprivation is due to the lack of deoxynucleotides needed for DNA synthesis. The lack of deoxynucleotides causes replicative stress leading to activation of the ataxia telangiectasia and Rad3-related protein (ATR)-mediated DNA damage pathway, which arrests cells in S-phase. The key metabolite generated from Q utilization was aspartate, which is generated from a transaminase reaction whereby Q-derived glutamate is converted to α-ketoglutarate with the concomitant conversion of oxaloacetate to aspartate. Aspartate is a critical metabolite for both purine and pyrimidine nucleotide biosynthesis. This study identifies the molecular basis for the S-phase arrest caused by Q deprivation in KRas-driven cancer cells that arrest in S-phase in response to Q deprivation. Given that arresting cells in S-phase sensitizes cells to apoptotic insult, this study suggests novel therapeutic approaches to KRas-driven cancers.

Consecutive incorporation of functionalized nucleotides with amphiphilic side chains by novel KOD polymerase mutant.

Recently, 7-substituted 7-deazapurine nucleoside triphosphates and 5-substituted pyrimidine nucleoside triphosphates (dN(am)TPs) were synthesized to extend enzymatically using commercially available polymerase. However, extension was limited when we attempted to incorporate the substrates consecutively. To address this, we have produced a mutant polymerase that can efficiently accept the modified nucleotide with amphiphilic groups as substrates. Here we show that the KOD polymerase mutant, KOD exo(-)/A485L, had the ability to incorporate dN(am)TP continuously over 50nt, indicating that the mutant is sufficient for generating functional nucleic acid molecules.

Pokeweed antiviral protein restores levels of cellular APOBEC3G during HIV-1 infection by depurinating Vif mRNA.

Pokeweed antiviral protein (PAP) is an RNA glycosidase that inhibits production of human immunodeficiency virus type 1 (HIV-1) when expressed in human culture cells. Previously, we showed that the expression of PAP reduced the levels of several viral proteins, including virion infectivity factor (Vif). However, the mechanism causing Vif reduction and the consequences of the inhibition were not determined. Here we show that the Vif mRNA is directly depurinated by PAP. Because of depurination at two specific sites within the Vif ORF, Vif levels decrease during infections and the progeny viruses that are generated are ∼ 10-fold less infectious and compromised for proviral integration. These results are consistent with PAP activity inhibiting translation of Vif, which in turn reduces the effect of Vif to inactivate the host restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like editing complex 3G). Our findings identify Vif mRNA as a new substrate for PAP and demonstrate that derepression of innate immunity against HIV-1 contributes to its antiviral activity.

Lie Markov models with purine/pyrimidine symmetry.

Continuous-time Markov chains are a standard tool in phylogenetic inference. If homogeneity is assumed, the chain is formulated by specifying time-independent rates of substitutions between states in the chain. In applications, there are usually extra constraints on the rates, depending on the situation. If a model is formulated in this way, it is possible to generalise it and allow for an inhomogeneous process, with time-dependent rates satisfying the same constraints. It is then useful to require that, under some time restrictions, there exists a homogeneous average of this inhomogeneous process within the same model. This leads to the definition of "Lie Markov models" which, as we will show, are precisely the class of models where such an average exists. These models form Lie algebras and hence concepts from Lie group theory are central to their derivation. In this paper, we concentrate on applications to phylogenetics and nucleotide evolution, and derive the complete hierarchy of Lie Markov models that respect the grouping of nucleotides into purines and pyrimidines-that is, models with purine/pyrimidine symmetry. We also discuss how to handle the subtleties of applying Lie group methods, most naturally defined over the complex field, to the stochastic case of a Markov process, where parameter values are restricted to be real and positive. In particular, we explore the geometric embedding of the cone of stochastic rate matrices within the ambient space of the associated complex Lie algebra.

Polypurine reverse Hoogsteen hairpins as a gene therapy tool against survivin in human prostate cancer PC3 cells in vitro and in vivo.

As a new approach for gene therapy, we recently developed a new type of molecule called polypurine reverse Hoogsteen hairpins (PPRHs). We decided to explore the in vitro and in vivo effect of PPRHs in cancer choosing survivin as a target since it is involved in apoptosis, mitosis and angiogenesis, and overexpressed in different tumors. We designed four PPRHs against the survivin gene, one of them directed against the template strand and three against different regions of the coding strand. These PPRHs were tested in PC3 prostate cancer cells in an in vitro screening of cell viability and apoptosis. PPRHs against the promoter sequence were the most effective and caused a decrease in survivin mRNA and protein levels. We confirmed the binding between the selected PPRHs and their target sequences in the survivin gene. In addition we determined that both the template- and the coding-PPRH targeting the survivin promoter were interfering with the binding of transcription factors Sp1 and GATA-3, respectively. Finally, we conducted two in vivo efficacy assays using the Coding-PPRH against the survivin promoter and performing two routes of administration, namely intratumoral and intravenous, in a subcutaneous xenograft tumor model of PC3 prostate cancer cells. The results showed that the chosen Coding-PPRH proved to be effective in decreasing tumor volume, and reduced the levels of survivin protein and the formation of blood vessels. These findings represent the preclinical proof of principle of PPRHs as a new silencing tool for cancer gene therapy.

Nucleotides adjacent to the ligand-binding pocket are linked to activity tuning in the purine riboswitch.

Direct sensing of intracellular metabolite concentrations by riboswitch RNAs provides an economical and rapid means to maintain metabolic homeostasis. Since many organisms employ the same class of riboswitch to control different genes or transcription units, it is likely that functional variation exists in riboswitches such that activity is tuned to meet cellular needs. Using a bioinformatic approach, we have identified a region of the purine riboswitch aptamer domain that displays conservation patterns linked to riboswitch activity. Aptamer domain compositions within this region can be divided into nine classes that display a spectrum of activities. Naturally occurring compositions in this region favor rapid association rate constants and slow dissociation rate constants for ligand binding. Using X-ray crystallography and chemical probing, we demonstrate that both the free and bound states are influenced by the composition of this region and that modest sequence alterations have a dramatic impact on activity. The introduction of non-natural compositions result in the inability to regulate gene expression in vivo, suggesting that aptamer domain activity is highly plastic and thus readily tunable to meet cellular needs.

Different characteristics and nucleotide binding properties of inosine monophosphate dehydrogenase (IMPDH) isoforms.

We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP) would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i) the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii) communication occurs between the Bateman and catalytic domains and (iii) the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

Gene expression profile of early in vitro biofilms of Streptococcus pneumoniae.

In this study, the gene expression profile of early in vitro Streptococcus pneumoniae biofilm with respect to planktonic cells in cDNA microarray analysis is reported. Microarray analysis with respect to planktonic cells was performed on total RNA extracted from biofilms grown in 24-well microtiter plates. To validate the microarray results, real-time RT-PCR was performed on 13 differentially expressed genes and one constitutively expressed gene. The cDNA-microarray analyses identified 89 genes that were significantly differentially expressed in biofilm and planktonic cells. Genes involved in isoprenoid biosynthesis, cell wall biosynthesis, translation and purine and pyrimidine nucleotide metabolic pathways were exclusively expressed in the biofilms, whereas transcription regulator genes were exclusively expressed in planktonic cells. The real-time RT-PCR results of 13 differentially regulated genes were completely in agreement with the microarray data. The exclusive up regulation in biofilms of genes involved in the mevalonate pathway, cell wall biosynthesis, translation and purine and pyrimidine nucleotide metabolic pathways suggests that expression of these genes may be required for initial biofilm formation, and growth and survival of bacteria in biofilms. The up regulation of related genes suggests that cells in biofilms may be under stress conditions and possibly actively involved in the protein synthesis required to adapt to a new environment.

The polypyrimidine/polypurine motif in the mouse mu opioid receptor gene promoter is a supercoiling-regulatory element.

The mu opioid receptor (MOR) is the principle molecular target of opioid analgesics. The polypyrimidine/polypurine (PPy/u) motif enhances the activity of the MOR gene promoter by adopting a non-B DNA conformation. Here, we report that the PPy/u motif regulates the processivity of torsional stress, which is important for endogenous MOR gene expression. Analysis by topoisomerase assays, S1 nuclease digests, and atomic force microscopy showed that, unlike homologous PPy/u motifs, the position- and orientation-induced structural strains to the mouse PPy/u element affect its ability to perturb the relaxation activity of topoisomerase, resulting in polypurine strand-nicked and catenated DNA conformations. Raman spectrum microscopy confirmed that mouse PPy/u containing-plasmid DNA molecules under the different structural strains have a different configuration of ring bases as well as altered Hoogsteen hydrogen bonds. The mouse MOR PPy/u motif drives reporter gene expression fortyfold more effectively in the sense orientation than in the antisense orientation. Furthermore, mouse neuronal cells activate MOR gene expression in response to the perturbations of topology by topoisomerase inhibitors, whereas human cells do not. These results suggest that, interestingly among homologous PPy/u motifs, the mouse MOR PPy/u motif dynamically responds to torsional stress and consequently regulates MOR gene expression in vivo.

Interaction of the diguanylate cyclase YdeH of Escherichia coli with 2',(3')-substituted purine and pyrimidine nucleotides.

Diguanylate cyclases (DGCs) synthesize the bacterial second messenger cyclic 3',5'-diguanosine monophosphate (c-di-GMP), which is degraded by specific phosphodiesterases. c-di-GMP levels control the transition of bacteria from a motile to a biofilm-forming lifestyle. These bacterial communities are highly resistant to antibiotic treatment and represent the predominant lifestyle in most chronic infections. Hence, DGCs serve as starting point for the development of novel therapeutics interfering with the second messenger-signaling network in bacteria. In previous studies, we showed that 2'(3')-O-(N-methylanthraniloyl) (MANT)- and 2',3'-O-(2,4,6-trinitrophenyl) (TNP)-substituted nucleotides are potent adenylyl and guanylyl cyclase inhibitors. The catalytic domain of DGCs is homologous to the mammalian adenylyl cyclase catalytic domain. Therefore, we investigated the interaction of various MANT purine and pyrimidine nucleotides with the model DGC YdeH from Escherichia coli. We observed strong fluorescence resonance energy transfer between tryptophan and tyrosine residues of YdeH and the MANT group of MANT-NTPs (MANT-ATP, -CTP, -GTP, -ITP, -UTP, and -XTP) and an enhanced direct MANT fluorescence upon interaction with YdeH. We assessed the affinity of MANT-NTPs to YdeH by performing competition assays with NTPs. We conducted an amino acid alignment of YdeH with the earlier crystallized Caulobacter crescentus DGC PleD and found high similarities in the nucleotide-binding site of PleD. In vitro mass-spectrometric activity assays with YdeH resulted in the identification of new MANT/TNP nucleotide-based inhibitors of DGC activity. Together, the analysis of interactions between MANT/TNP nucleotides and YdeH provides a new basis for the identification and development of DGC inhibitors and allows insights into nucleotide-protein interactions.

Cross-linking to an interrupted polypurine sequence with a platinum-modified triplex-forming oligonucleotide.

Triplex-forming oligonucleotides (TFOs) can bind specifically to polypurine sequences in double-stranded DNA. A single interruption of this polypurine tract can greatly destabilize triplex formation. The stability of triplexes can be significantly enhanced by covalently linking the TFO to its DNA target with reactive functional groups conjugated to the TFO. Covalently cross-linked TFOs are effective inhibitors of transcription of the target DNA sequence. We have designed a TFO with a platinum-modified base that can interact with and cross-link to a cytosine interruption in the polypurine tract of a target DNA duplex. The TFO contains an N(4)-(aminoalkyl)cytosine derivatized with cis-diamminediaquaplatinum(II) or trans-diamminediaquaplatinum(II). When bound to its target, the tethered platinum of the TFO can reach across the major groove and form an adduct with the guanine N7 of the interrupting C.G base pair. The optimal tether length is five methylene groups, and cross-linking is most efficient when the tether is modified with trans-diamminediaquaplatinum(II). Cross-linking requires that the TFO is bound to its designated DNA target. Addition of cyanide to the cross-linked TFO product reversed the cross-link, behavior that is consistent with the presence of a platinum-guanine adduct. The kinetics of the cross-linking reaction were studied and the half-life of the cross-linking reaction was approximately 3 h. Our results demonstrate that platinum-conjugated TFOs can be designed to cross-link with DNA targets that contain a single pyrimidine interruption. Modifications of this type may prove useful for expanding the DNA sequences that can be targeted by TFOs and increasing the stability of the resulting triplexes.

The phosphatidylinositol 3-kinase/akt cassette regulates purine nucleotide synthesis.

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is highly conserved throughout evolution and regulates cell size and survival and cell cycle progression. It regulates the latter by stimulating procession through G(1) and the G(1)/S phase transition. Entry into S phase requires an abundant supply of purine nucleotides, but the effect of the PI3K/Akt pathway on purine synthesis has not been studied. We now show that the PI3K/Akt cassette regulates both de novo and salvage purine nucleotide synthesis in insulin-responsive mouse mesenchymal cells. We found that serum and insulin stimulated de novo purine synthesis in serum-starved cells largely through PI3K/Akt signaling, and pharmacologic and genetic inhibition of PI3K/Akt reduced de novo synthesis by 75% in logarithmically growing cells. PI3K/Akt regulated early steps of de novo synthesis by modulating phosphoribosylpyrophosphate production by the non-oxidative pentose phosphate pathway and late steps by modulating activity of the bifunctional enzyme aminoimidazole-carboxamide ribonucleotide transformylase IMP cyclohydrolase, an enzyme not previously known to be regulated. The effects of PI3K/Akt on purine nucleotide salvage were likely through regulating phosphoribosylpyrophosphate availability. These studies define a new mechanism whereby the PI3K/Akt cassette functions as a master regulator of cellular metabolism and a key player in oncogenesis.

Strong short-range correlations and dichotomic codon classes in coding DNA sequences.

The study of correlation structures in DNA sequences is of great interest because it allows us to obtain structural and functional information about underlying genetic mechanisms. In this paper we present a study of the correlation structure of protein coding sequences of DNA based on a recently developed mathematical representation of the genetic code. A fundamental consequence of such representation is that codons can be assigned a parity class (odd-even). Such parity can be obtained by means of a nonlinear algorithm acting on the chemical character of the codon bases. In the same setting the Rumer's class can be naturally described and a new dichotomic class, the hidden class, can be defined. Moreover, we show that the set of DNA's base transformations associated to the three dichotomic classes can be put in a compact group-theoretic framework. We use the dichotomic classes as a coding scheme for DNA sequences and study the mutual dependence between such classes. The same analysis is carried out also on the chemical dichotomies of DNA bases. In both cases, the statistical analysis is performed by using an entropy-based dependence metric possessing many desirable properties. We obtain meaningful tests for mutual dependence by using suitable resampling techniques. We find strong short-range correlations between certain combinations of dichotomic codon classes. These results support our previous hypothesis that codon classes might play an active role in the organization of genetic information.

Dysregulation of purine nucleotide biosynthesis pathways modulates cisplatin cytotoxicity in Saccharomyces cerevisiae.

We found previously that inactivation of the FCY2 gene, encoding a purine-cytosine permease, or the HPT1 gene, encoding the hypoxanthine guanine phosphoribosyl transferase, enhances cisplatin resistance in yeast cells. Here, we report that in addition to fcy2Delta and hpt1Delta mutants in the salvage pathway of purine nucleotide biosynthesis, mutants in the de novo pathway that disable the feedback inhibition of AMP and GMP biosynthesis also enhanced cisplatin resistance. An activity-enhancing mutant of the ADE4 gene, which constitutively synthesizes AMP and excretes hypoxanthine, and a GMP kinase mutant (guk1), which accumulates GMP and feedback inhibits Hpt1 function, both enhanced resistance to cisplatin. In addition, overexpression of the ADE4 gene in wild-type cells, which increases de novo synthesis of purine nucleotides, also resulted in elevated cisplatin resistance. Cisplatin cytotoxicity in wild-type cells was abolished by low concentration of extracellular purines (adenine, hypoxanthine, and guanine) but not cytosine. Inhibition of cytotoxicity by exogenous adenine was accompanied by a reduction of DNA-bound cisplatin in wild-type cells. As a membrane permease, Fcy2 may mediate limited cisplatin transport because cisplatin accumulation in whole cells was slightly affected in the fcy2Delta mutant. However, the fcy2Delta mutant had a greater effect on the amount of DNA-bound cisplatin, which decreased to 50 to 60% of that in the wild-type cells. Taken together, our results indicate that dysregulation of the purine nucleotide biosynthesis pathways and the addition of exogenous purines can modulate cisplatin cytotoxicity in Saccharomyces cerevisiae.