Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor.
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Deoxyhypusine synthase catalyses the first step in the post-translational synthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl) lysine] in a single cellular protein, the precursor of eukaryotic initiation factor 5A (eIF5A). Deoxyhypusine synthase exists as a tetramer with four potential active sites. The formation of a stable complex between human deoxyhypusine synthase and its protein substrate, human recombinant eIF5A precursor (ec-eIF5A), was examined by affinity chromatography using polyhistidine-tagged (His.Tag) ec-eIF5A, by a gel mobility-shift method, and by analytical ultracentrifugation. Deoxyhypusine synthase was selectively retained by His.Tag-ec-eIF5A immobilized on a resin. The complex of deoxyhypusine synthase and ec-eIF5A was separated from the free enzyme and protein substrate by electrophoresis under non-denaturing conditions. The stoichiometry of the two components in the complex was estimated to be 1 deoxyhypusine synthase tetramer to 1 ec-eIF5A monomer by N-terminal amino acid sequencing of the complex. Equilibrium ultracentrifugation data further supported this 1:1 ratio and indicated a very strong interaction of the enzyme with ec-eIF5A (Kd+info)
Can G-C Hoogsteen-wobble pairs contribute to the stability of d(G. C-C) triplexes?
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Quantum mechanics, molecular dynamics and statistical mechanics methods are used to analyze the importance of neutral Hoogsteen-wobble G.C pairing in the stabilization of triple helices based on the poly-(G.C-C) trio at neutral pH and low ionic strength. In spite of the existence of a single hydrogen bond, the Hoogsteen-wobble G.C pair is found to be quite stable both in gas phase and solvated DNA. Molecular dynamics simulations of different triplexes based on the d(G.C-C) trio leads to stable structures if the neutral d(G.C-C) steps stabilized by Hoogsteen-wobble pairs are mixed with d(G.C-C+) steps. Finally, high level ab initio calculations and thermodynamic integration techniques are used to determine the relative stability of G.C wobble and G.C imino pairings. It is found that triplexes containing the imino pairing are slightly more stable structures than those with the wobble one, due mainly to a better stacking. (+info)
The type of DNA glycosylase determines the base excision repair pathway in mammalian cells.
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The base excision repair (BER) of modified nucleotides is initiated by damage-specific DNA glycosylases. The repair of the resulting apurinic/apyrimidinic site involves the replacement of either a single nucleotide (short patch BER) or of several nucleotides (long patch BER). The mechanism that controls the selection of either BER pathway is unknown. We tested the hypothesis that the type of base damage present on DNA, by determining the specific DNA glycosylase in charge of its excision, drives the repair of the resulting abasic site intermediate to either BER branch. In mammalian cells hypoxanthine (HX) and 1,N6-ethenoadenine (epsilonA) are both substrates for the monofunctional 3-methyladenine DNA glycosylase, the ANPG protein, whereas 7,8-dihydro-8-oxoguanine (8-oxoG) is removed by the bifunctional DNA glycosylase/beta-lyase 8-oxoG-DNA gly- cosylase (OGG1). Circular plasmid molecules containing a single HX, epsilonA, or 8-oxoG were constructed. In vitro repair assays with HeLa cell extracts revealed that HX and epsilonA are repaired via both short and long patch BER, whereas 8-oxoG is repaired mainly via the short patch pathway. The preferential repair of 8-oxoG by short patch BER was confirmed by the low efficiency of repair of this lesion by DNA polymerase beta-deficient mouse cells as compared with their wild-type counterpart. These data fit into a model where the intrinsic properties of the DNA glycosylase that recognizes the lesion selects the branch of BER that will restore the intact DNA template. (+info)
Vps9p is a guanine nucleotide exchange factor involved in vesicle-mediated vacuolar protein transport.
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Vacuolar protein sorting (vps) mutants of Saccharomyces cerevisiae missort and secrete vacuolar hydrolases. The gene affected in one of these mutants, VPS21, encodes a member of the Sec4/Ypt/Rab family of small GTPases. Rab proteins play an essential role in vesicle-mediated protein transport. Using both yeast two-hybrid assays and chemical cross-linking, we have identified another VPS gene product, Vps9p, that preferentially interacts with a mutant form of Vps21p-S21N that binds GDP but not GTP. In vitro purified Vps9p was found to stimulate GDP release from Vps21p in a dose-dependent manner. Vps9p also stimulated GTP association as a result of facilitated GDP release. However, Vps9p did not stimulate guanine nucleotide exchange of GTP-bound Vps21p or GTP hydrolysis. We tested the ability of Vps9p to stimulate the intrinsic guanine nucleotide exchange activity of Rab5, which is a mammalian sequence homologue of Vps21p, and Ypt7p, which is another yeast Rab protein involved in vacuolar protein transport. Rab5, but not Ypt7p was responsive to Vps9p, which indicates that Vps9p recognizes sequence variation among Rab proteins. We conclude that Vps9p is a novel guanine nucleotide exchange factor that is specific for Vps21p/Rab5. Since there are no obvious Vps9p sequence homologues in yeast, Vps9p may also possess unique regulatory functions required for vacuolar protein transport. (+info)
Analysis of 8-hydroxyguanine in rat kidney genomic DNA after administration of a renal carcinogen, ferric nitrilotriacetate.
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The frequency of oxidative base damage, such as 8-hydroxyguanine (8-OH-Gua), was determined at the nucleotide level of resolution using the ligation-mediated PCR technique. Administration of a renal carcinogen, ferric nitrilotriacetate (Fe-NTA), is known to induce oxidative stress and subsequent formation of 8-OH-Gua in the rat kidney. Whole genomic DNA was isolated from the rat kidney after or without Fe-NTA treatment and then cleaved with hot piperidine. In order to assess the frequency of 8-OH-Gua formation, we chose three genes, the tumor suppressor gene p53, the heat shock protein 70 (HSP70-1) gene and the Na,K-ATPase alpha1 subunit gene. No alteration in the cleavage profile was observed in the p53 and HSP70 genes after Fe-NTA treatment. In the case of the p53 gene, a low incidence of point mutations has been observed in this carcinogenesis system. On the other hand, time-dependent alterations, corresponding to the time course of overall 8-OH-Gua formation and repair, were detected in the promoter region of the Na,K-ATPase alpha1 subunit gene. GpG and GpGpG in specific regions seem to be hotspots for the formation of 8-OH-Gua. These results were confirmed by formamidopyrimidine-DNA glycosylase-dependent DNA cleavage patterns. Thus, oxidative base damage, such as 8-OH-Gua, was not distributed uniformly along the whole genome, but seemed to be restricted to particular genes and regions. (+info)
G+C content variation along and among Saccharomyces cerevisiae chromosomes.
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Past analyses of the genome of the yeast Saccharomyces cerevisiae have revealed substantial regional variation in G+C content. Important questions remain, though, as to the origin, nature, significance, and generality of this variation. We conducted an extensive analysis of the yeast genome to try to answer these questions. Our results indicate that open reading frames (ORFs) with similar G+C contents at silent codon positions are significantly clustered on chromosomes. This clustering can be explained by very short range correlations of silent-site G+C contents at neighboring ORFs. ORFs of high silent-site G+C content are disproportionately concentrated on shorter chromosomes, which causes a negative relationship between chromosome length and G+C content. Contrary to previous reports, there is no correlation between gene density and silent-site G+C content in yeast. Chromosome III is atypical in many regards, and possible reasons for this are discussed. (+info)
High affinity interactions of nucleolin with G-G-paired rDNA.
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Nucleolin is a very abundant eukaryotic protein that localizes to the nucleolus, where the rDNA undergoes transcription, replication, and recombination and where rRNA processing occurs. The top (non-template) strand of the rDNA is very guanine-rich and has considerable potential to form structures stabilized by G-G pairing. We have assayed binding of endogenous and recombinant nucleolin to synthetic oligonucleotides in which G-rich regions have formed intermolecular G-G pairs to produce either two-stranded G2 or four-stranded G4 DNA. We report that nucleolin binds G-G-paired DNA with very high affinity; the dissociation constant for interaction with G4 DNA is KD = 1 nM. Two separate domains of nucleolin can interact with G-G-paired DNA, the four RNA binding domains and the C-terminal Arg-Gly-Gly repeats. Both domains bind G4 DNA with high specificity and recognize G4 DNA structure independent of sequence context. The high affinity of the nucleolin/G4 DNA interaction identifies G-G-paired structures as natural binding targets of nucleolin in the nucleolus. The ability of two independent domains of nucleolin to bind G-G-paired structures suggests that nucleolin can function as an architectural factor in rDNA transcription, replication, or recombination. (+info)
O6-benzylguanine: a clinical trial establishing the biochemical modulatory dose in tumor tissue for alkyltransferase-directed DNA repair.
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Early phase evaluation of anticancer drugs has traditionally used toxicity (usually hematological) rather than efficacy end points to establish appropriate dosing schedules. To establish a biochemical efficacy end point for overcoming alkylguanine DNA alkyltransferase (AGT)-mediated tumor cell resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea, we performed a novel dose escalation clinical trial for the AGT-depleting agent O6-benzylguanine (BG). The dose of BG required to deplete AGT to undetectable levels (BMD(T)) in sequential computed tomography-guided tumor tissue biopsies before BG and 18 h after BG was determined. Thirty patients received doses of BG ranging from 10 to 120 mg/m2. In tumor tissue, AGT depletion >86% of baseline was demonstrated at all doses tested. Residual tumor AGT activity, present 18 h after BG doses of 10-80 mg/m2, was eliminated at the 120 mg/m2 dose and is thus the BMD(T) of BG. BG pharmacokinetics are characterized by the rapid, dose-independent clearance of BG from plasma Metabolism of BG to its biologically active metabolite, 8-oxo-benzylguanine (8-oxo-BG), was found. The t(1/2) of 8-oxo-BG is longer than BG. Plasma concentrations of 8-oxo-BG well above 200 ng/ml 18 h after the end of the BG infusion were observed at the highest dose levels tested and appeared to correlate with depletion of AGT activity to undetectable levels in tumor tissue. AGT activity in peripheral blood mononuclear cells at baseline did not correlate with tumor tissue AGT activity. Depletion of AGT activity to undetectable levels in peripheral blood mononuclear cells occurred at lower doses and was not a reliable predictor for tumor tissue depletion. No serious side effects were observed with administration of BG alone or in combination with 13 mg/m2 1,3-bis(2-chloroethyl)-1-nitrosourea. This is the first clinical study in which biochemical analyses from pre- and posttreatment tumor biopsies have been used as an efficacy end point for the clinical development of an anticancer agent. From our tumor tissue biopsy data, we have established that a BG dose of 120 mg/m2 infused over 1 h should be used in Phase II clinical trials. (+info)