Identification of O-acetylserine(thiol)lyase (OASTL) genes in sorghum (Sorghum bicolor) and gene expression analysis under cadmium stress.

Cysteine (Cys) is the first identified molecule in plant metabolism which includes both sulfur and nitrogen. It can be synthesized in three cellular compartments, containing chloroplast, cytoplasm and mitochondrion. The final step of cysteine biosynthesis is catalyzed by the O-acetylserine(thiol)lyase enzyme (OASTL, E.C. 4.2.99). In the present study, seven members of the OASTL gene family in the sorghum (Sorghum bicolor) genome were identified at a genome-wide scale and comparative bioinformatics analyses were performed between sorghum and Arabidopsis OASTLs. In all OASTL proteins, a pyridoxal-phosphate dependent domain structure (PALP, PF00291) was identified. The gene ontology annotations also revealed that all sorghum OASTL genes have KOG1252 (Cystathionine beta-synthase and related enzyme) and K01738 (cysteine synthase A) activities. In promotor sequences of OASTL genes, diverse cis-acting elements were found, including hormone and light responsiveness, abiotic stress responsiveness, and tissue-specific ones (meristem and endosperm). Sorghum OASTL genes demonstrated medium or high level expressions in anatomical parts and developmental stages based on the digital expression data. Expression of OASTL genes were also analyzed under cadmium (Cd) stress in sorghum by Real Time-quantitative PCR (RT-qPCR). The results exclusively showed that OASTL A1-2 gene was 1.12 fold up-regulated in roots, whereas cysteine synthase 26 was 2.25 fold down-regulated in leaves. The predicted 3D structure of OASTLs indicated some structural diversities as well as variations in the secondary structures.

Activation of R-mediated innate immunity and disease susceptibility is affected by mutations in a cytosolic O-acetylserine (thiol) lyase in Arabidopsis.

O-acetylserine (thiol) lyases (OASTLs) are evolutionarily conserved proteins among many prokaryotes and eukaryotes that perform sulfur acquisition and synthesis of cysteine. A mutation in the cytosolic OASTL-A1 protein ONSET OF LEAF DEATH3 (OLD3) was previously shown to reduce the OASTL activity of the old3-1 protein in vitro and cause auto-necrosis in specific Arabidopsis accessions. Here we investigated why a mutation in this protein causes auto-necrosis in some but not other accessions. The auto-necrosis was found to depend on Recognition of Peronospora Parasitica 1 (RPP1)-like disease resistance R gene(s) from an evolutionarily divergent R gene cluster that is present in Ler-0 but not the reference accession Col-0. RPP1-like gene(s) show a negative epistatic interaction with the old3-1 mutation that is not linked to reduced cysteine biosynthesis. Metabolic profiling and transcriptional analysis further indicate that an effector triggered-like immune response and metabolic disorder are associated with auto-necrosis in old3-1 mutants, probably activated by an RPP1-like gene. However, the old3-1 protein in itself results in largely neutral changes in primary plant metabolism, stress defence and immune responses. Finally, we showed that lack of a functional OASTL-A1 results in enhanced disease susceptibility against infection with virulent and non-virulent Pseudomonas syringae pv. tomato DC3000 strains. These results reveal an interaction between the cytosolic OASTL and components of plant immunity.

Regulatory role of cystathionine-gamma-synthase and de novo synthesis of methionine in ethylene production during tomato fruit ripening.

The essential amino acid methionine is a substrate for the synthesis of S-adenosyl-methionine (SAM), that donates its methyl group to numerous methylation reactions, and from which polyamines and ethylene are generated. To study the regulatory role of methionine synthesis in tomato fruit ripening, which requires a sharp increase in ethylene production, we cloned a cDNA encoding cystathionine gamma-synthase (CGS) from tomato and analysed its mRNA and protein levels during tomato fruit ripening. CGS mRNA and protein levels peaked at the "turning" stage and declined as the fruit ripened. Notably, the tomato CGS mRNA level in both leaves and fruit was negatively affected by methionine feeding, a regulation that Arabidopsis, but not potato CGS mRNA is subject to. A positive correlation was found between elevated ethylene production and increased CGS mRNA levels during the ethylene burst of the climacteric ripening of tomato fruit. In addition, wounding of pericarp from tomato fruit at the mature green stage stimulated both ethylene production and CGS mRNA level. Application of exogenous methionine to pericarp of mature green fruit increased ethylene evolution, suggesting that soluble methionine may be a rate limiting metabolite for ethylene synthesis. Moreover, treatment of mature green tomato fruit with the ethylene-releasing reagent Ethephon caused an induction of CGS mRNA level, indicating that CGS gene expression is regulated by ethylene. Taken together, these results imply that in addition to recycling of the methionine moieties via the Yang pathway, operating during synthesis of ethylene, de novo synthesis of methionine may be required when high rates of ethylene production are induced.

Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum.

Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite D,L-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of L-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.

Stat3 protects against Fas-induced liver injury by redox-dependent and -independent mechanisms.

Signal transducer and activator of transcription-3 (Stat3) is one of the most important molecules involved in the initiation of liver development and regeneration. In order to investigate the hepatoprotective effects of Stat3, we examined whether Stat3 protects against Fas-mediated liver injury in the mouse. A constitutively activated form of Stat3 (Stat3-C) was adenovirally overexpressed in mouse liver by intravenous injection, and then a nonlethal dose of Fas agonist (Jo2) was injected intraperitoneally into the mouse (0.3 microg/g body wt). Stat3-C dramatically suppressed both apoptosis and necrosis induced by Jo2. In contrast, liver-specific Stat3-knockout mice failed to survive following Jo2 injection. Stat3-C upregulated expression of FLICE inhibitor protein (FLIP), Bcl-xL, and Bcl-2, and accordingly downregulated activities of FLICE and caspase-3 that were redox-independent. Interestingly, Stat3-C also upregulated the redox-associated protein redox factor-1 (Ref-1) and reduced apoptosis in liver following Jo2 injection by suppressing oxidative stress and redox-sensitive caspase-3 activity. These findings indicate that Stat3 activation protects against Fas-mediated liver injury by inhibiting caspase activities in redox-dependent and -independent mechanisms.

Base excision repair activities required for yeast to attain a full chronological life span.

The chronological life span of yeast, the survival of stationary (G0) cells over time, provides a model for investigating certain of the factors that may influence the aging of non-dividing cells and tissues in higher organisms. This study measured the effects of defined defects in the base excision repair (BER) system for DNA repair on this life span. Stationary yeast survives longer when it is pre-grown on respiratory, as compared to fermentative (glucose), media. It is also less susceptible to viability loss as the result of defects in DNA glycosylase/AP lyases (Ogg1p, Ntg1p, Ntg2p), apurinic/apyrimidinic (AP) endonucleases (Apn1p, Apn2p) and monofunctional DNA glycosylase (Mag1p). Whereas single BER glycosylase/AP lyase defects exerted little influence over such optimized G0 survival, this survival was severely shortened with the loss of two or more such enzymes. Equally, the apn1delta and apn2delta single gene deletes survived as well as the wild type, whereas a apn1delta apn2delta double mutant totally lacking in any AP endonuclease activity survived poorly. Both this shortened G0 survival and the enhanced mutagenicity of apn1delta apn2delta cells were however rescued by the over-expression of either Apn1p or Apn2p. The results highlight the vital importance of BER in the prevention of mutation accumulation and the attainment of the full yeast chronological life span. They also reveal an appreciable overlap in the G0 maintenance functions of the different BER DNA glycosylases and AP endonucleases.

Suppressive activities of OGG1 and MYH proteins against G:C to T:A mutations caused by 8-hydroxyguanine but not by benzo[a]pyrene diol epoxide in human cells in vivo.

8-Hydroxyguanine (8OHG), an oxidatively damaged base, and benzo[a]pyrene-diol-epoxide (BPDE), a metabolite of benzo[a]pyrene found in cigarette smoke, are thought to be major causes for G:C to T:A transversions in DNA of human cells. In this study, we assessed the abilities of OGG1, MYH and APE1 proteins, which are components of a base excision repair pathway, to suppress G:C to T:A transversions caused by 8OHG or BPDE by a bacterial suppressor tRNA (supF) forward mutation assay using a shuttle plasmid, pMY189. The introduction of a single 8OHG residue at position 159 of the supF gene and treatment with BPDE led to a 65- and 34-fold increase in mutation frequencies of the pMY189 plasmid, respectively, after replication in the NCI-H1299 human lung cancer cell line. G:C to T:A transversions were predominantly induced in these plasmids. Both the mutation frequency of the 8OHG-containing plasmid in NCI-H1299 cells and the occurrence of G:C to T:A transversions at position 159 in the supF gene were significantly reduced by overexpression of OGG1 and MYH proteins, but not by that of APE1 protein. In contrast, neither mutation frequency nor the occurrence of G:C to T:A transversion of the BPDE-treated plasmid was reduced by overexpression of OGG1, MYH and APE1 proteins. These results indicate that OGG1 and MYH function as suppressors for G:C to T:A transversions by 8OHG but not by BPDE in human cells.

Role of the AP-1 element and redox factor-1 (Ref-1) in mediating transcriptional induction of DT-diaphorase gene expression by oltipraz: a target for chemoprevention.

The dithiolethione oltipraz is a potent chemopreventive agent in preclinical models, and induces the expression of protective enzymes in the colon mucosa and peripheral mononuclear cells of treated human subjects. We investigated the effects of oltipraz on DT-diaphorase expression in HT29 colon adenocarcinoma cells. Following a 24-hr exposure to 100 microM oltipraz, elevated steady-state levels of mRNA for Jun and Fos family members were observed. A nuclear run-on assay showed induction of c-fos and c-jun transcripts at the end of the exposure, peaking at 12 hr after resuspension of cells in drug-free medium. Gel mobility shift analysis revealed a similar time-course of induced nuclear factor binding to an AP-1 probe. Supershift analysis verified the participation of Jun and Fos in the complexes. The redox coactivator Ref-1, a function of which is to enhance AP-1 binding, was induced 5-fold by oltipraz. Immunodepletion of Ref-1 partially inhibited factor binding to the AP-1 probe. Deletion analysis of the DT-diaphorase promoter in a CAT reporter construct revealed that loss of the AP-1 site accounted for approximately 65% of the induction by oltipraz. Mutation of the AP-1 element in a full-length promoter construct yielded similar results. These data suggest the importance of transcriptional activation mediated by AP-1 in the chemopreventive activity of oltipraz, and indicate that novel chemoprevention structures may be selected based upon agonist activity at this locus.

The exonuclease activity of human apurinic/apyrimidinic endonuclease (APE1). Biochemical properties and inhibition by the natural dinucleotide Gp4G.

Human DNA apurinic/apyrimidinic endonuclease (APE1) plays a key role in the DNA base excision repair process. In this study, we further characterized the exonuclease activity of APE1. The magnesium requirement and pH dependence of the exonuclease and endonuclease activities of APE1 are significantly different. APE1 showed a similar K(m) value for matched, 3' mispaired, or nucleoside analog beta-l-dioxolane-cytidine terminated nicked DNA as well as for DNA containing a tetrahydrofuran, an abasic site analog. The k(cat) for exonuclease activity on matched, 3' mispaired, and beta-l-dioxolane-cytidine nicked DNA are 2.3, 61.2, and 98.8 min(-1), respectively, and 787.5 min(-1) for APE1 endonuclease. Site-directed APE1 mutant proteins (E96A, E96Q, D210E, D210N, and H309N), which target amino acid residues in the endonuclease active site, also showed significant decrease in exonuclease activity. Gp(4)G was the only potent inhibitor to compete against the substrates of endonuclease and exonuclease activities among all tested naturally occurring ribo-, deoxyribo-nucleoside/nucleotides, NAD(+), NADP(+), and Ap(4)A. The K(i) values of Gp(4)G for the endonuclease and exonuclease activities of APE1 are 10 +/- 0.6 and 1 +/- 0.2 microm, respectively. Given the relative concentrations of Gp(4)G, 3' mispaired, and abasic DNA, Gp(4)G may play an important role in regulating APE1 activity in cells. The data presented here suggest that the APE1 exonuclease and AP endonuclease are two distinct activities. APE1 may exist in two different conformations, and each conformation has a preference for a substrate. The different conformations can be affected by MgCl(2) or salt concentrations.

Implication of Ref-1 in the repression of renin gene transcription by intracellular calcium.

OBJECTIVE: The production of renin, which catalyzes the rate-limiting step of the renin-angiotensin system, is tightly regulated by intracellular second messengers. Among them, an increase of intracellular calcium represses renin gene expression. This inhibition of gene expression by intracellular calcium is exceptional, and the molecular mechanism supporting this phenomenon has not yet been identified. As the renin gene is negatively regulated by calcium in the same way as the parathormone (PTH) gene, we hypothesized that a similar molecular transcriptional mechanism could be involved. RESULTS: Analysis of the human renin proximal promoter led to the identification of a negative calcium response element (nCaRE), which is identical to the region of the PTH promoter and is involved in its repression by calcium. Transfection experiments in renin-expressing chorio-decidual cells demonstrated the transcriptional functionality of the human renin promoter nCaRE. In addition, mutation of nCaRE suppressed the sensitivity of the renin promoter to the increase in intracellular calcium. Gel shift assays demonstrated that Redox factor 1, a multifunctional protein involved in the repair of damaged DNA and the redox activation of AP-1 transcriptional factors, binds specifically to nCaRE. Immunostaining showed that this factor is translocated from the cytoplasm to the nucleus in response to an increase in the intracellular calcium concentration. CONCLUSION: Thus, the repression of renin expression by intracellular calcium may be mediated by the calcium-induced translocation of Ref-1 to the nucleus, where it binds to the renin promoter nCaRE, to repress the transcription of the renin gene.

The apurinic/apyrimidinic endonuclease activity of Ape1/Ref-1 contributes to human glioma cell resistance to alkylating agents and is elevated by oxidative stress.

Alkylating agents are standard components of adjuvant chemotherapy for gliomas. We provide evidence here that Ape1/Ref-1, the major mammalian apurinic/apyrimidinic endonuclease (Ap endo), contributes to alkylating agent resistance in human glioma cells by incising DNA at abasic sites. We show that antisense oligonucleotides directed against Ape1/Ref-1 in SNB19, a human glioma cell line lacking O(6)-methylguanine-DNA-methyltransferase, mediate both reduction in Ape1/Ref-1 protein and Ap endo activity and concurrent reduction in resistance to methyl methanesulfonate and the clinical alkylators temozolomide and 1,3-(2-chloroethyl)-1-nitrosourea. An accompanying increase in the level of abasic sites indicates that the DNA repair activity of Ape1/Ref-1 contributes to resistance. Conversely, we also show that exposure of SNB19 cells to HOCl, a generator of reactive oxygen species (ROS), results in elevated Ape1/Ref-1 protein and Ap endo activity, enhanced alkylator resistance, and reduced levels of abasic sites. Given current evidence that heightened oxidative stress prevails within brain tumors, the finding that ROS increase resistance to clinical alkylators in glioma cells may have significance for the response of gliomas to alkylating agent-based chemotherapy. Our results may also be relevant to the design of therapeutic regimens using concurrent ionizing radiation (a generator of ROS) and alkylating agent-based chemotherapy.

AP endonuclease 1 coordinates flap endonuclease 1 and DNA ligase I activity in long patch base excision repair.

Base loss is common in cellular DNA, resulting from spontaneous degradation and enzymatic removal of damaged bases. Apurinic/apyrimidinic (AP) endonucleases recognize and cleave abasic (AP) sites during base excision repair (BER). APE1 (REF1, HAP1) is the predominant AP endonuclease in mammalian cells. Here we analyzed the influences of APE1 on the human BER pathway. Specifically, APE1 enhanced the enzymatic activity of both flap endonuclease1 (FEN1) and DNA ligase I. FEN1 was stimulated on all tested substrates, regardless of flap length. Interestingly, we have found that APE1 can also inhibit the activities of both enzymes on substrates with a tetrahydrofuran (THF) residue on the 5'-downstream primer of a nick, simulating a reduced abasic site. However once the THF residue was displaced at least a single nucleotide, stimulation of FEN1 activity by APE1 resumes. Stimulation of DNA ligase I required the traditional nicked substrate. Furthermore, APE1 was able to enhance overall product formation in reconstitution of BER steps involving FEN1 cleavage followed by ligation. Overall, APE1 both stimulated downstream components of BER and prevented a futile cleavage and ligation cycle, indicating a far-reaching role in BER.

Targeted deletion of mNth1 reveals a novel DNA repair enzyme activity.

DNA N-glycosylase/AP (apurinic/apyrimidinic) lyase enzymes of the endonuclease III family (nth in Escherichia coli and Nth1 in mammalian organisms) initiate DNA base excision repair of oxidized ring saturated pyrimidine residues. We generated a null mouse (mNth1(-/-)) by gene targeting. After almost 2 years, such mice exhibited no overt abnormalities. Tissues of mNth1(-/-) mice contained an enzymatic activity which cleaved DNA at sites of oxidized thymine residues (thymine glycol [Tg]). The activity was greater when Tg was paired with G than with A. This is in contrast to Nth1, which is more active against Tg:A pairs than Tg:G pairs. We suggest that there is a back-up mammalian repair activity which attacks Tg:G pairs with much greater efficiency than Tg:A pairs. The significance of this activity may relate to repair of oxidized 5-methyl cytosine residues (5meCyt). It was shown previously (S. Zuo, R. J. Boorstein, and G. W. Teebor, Nucleic Acids Res. 23:3239-3243, 1995) that both ionizing radiation and chemical oxidation yielded Tg from 5meCyt residues in DNA. Thus, this previously undescribed, and hence novel, back-up enzyme activity may function to repair oxidized 5meCyt residues in DNA while also being sufficient to compensate for the loss of Nth1 in the mutant mice, thereby explaining the noninformative phenotype.

Stimulation of 3'-->5' exonuclease and 3'-phosphodiesterase activities of yeast apn2 by proliferating cell nuclear antigen.

The Apn2 protein of Saccharomyces cerevisiae contains 3'-->5' exonuclease and 3'-phosphodiesterase activities, and these activities function in the repair of DNA strand breaks that have 3'-damaged termini and which are formed in DNA by the action of oxygen-free radicals. Apn2 also has an AP endonuclease activity and functions in the removal of abasic sites from DNA. Here, we provide evidence for the physical and functional interaction of Apn2 with proliferating cell nuclear antigen (PCNA). As indicated by gel filtration and two-hybrid studies, Apn2 interacts with PCNA both in vitro and in vivo and mutations in the consensus PCNA-binding motif of Apn2 abolish this interaction. Importantly, PCNA stimulates the 3'-->5' exonuclease and 3'-phosphodiesterase activities of Apn2. We have examined the involvement of the interdomain connector loop (IDCL) and of the carboxy-terminal domain of PCNA in Apn2 binding and found that Apn2 binds PCNA via distinct domains dependent upon whether the binding is in the absence or presence of DNA. In the absence of DNA, Apn2 binds PCNA through its IDCL domain, whereas in the presence of DNA, when PCNA has been loaded onto the template-primer junction by replication factor C, the C-terminal domain of PCNA mediates the binding.

Redox factor-1/APE suppresses oxidative stress by inhibiting the rac1 GTPase.

Oxidative stress triggered by many environmental and clinical insults results in cellular injury and death. The small GTPase rac1 promotes oxidative stress via the production of reactive oxygen species (ROS). In turn, the homeostatic response to such stress includes up-regulation of the dual function reducing protein/DNA repair enzyme APE/redox factor-1(ref-1). In this report we explore the function and relationship between ref-1 and rac1 in the setting of oxidative stress triggered by re-oxygenation/reperfusion. In a model of mouse hepatic ischemia/reperfusion (I/R), recombinant adenoviral overexpression of ref-1 resulted in suppression of reperfusion-stimulated oxidative stress, NF-kB induction, apoptosis, and acute injury, whereas down-regulation of endogenous ref-1 by adenoviral expression of antisense ref-1 led to an increase in these reperfusion-induced parameters. Ref-1 also mitigated ROS production induced by adenoviral expression of an active form of rac1. Finally, overexpression of ref-1 in primary hepatocytes suppressed reoxygenation-stimulated rac1 activity. This work demonstrates a novel function of ref-1 in inhibition of rac1 activity, and rac1-mediated oxidative stress and injury.

Evidence for multiple imino intermediates and identification of reactive nucleophiles in peptide-catalyzed beta-elimination at abasic sites.

Prior investigations have demonstrated that peptides containing a single aromatic residue flanked by basic ones, such as Lys-Trp-Lys, can incise the phosphodiester backbone of duplex DNA at an AP site via beta-elimination. An amine serves as the reactive nucleophile to attack C1' on the ring-open deoxyribose sugar to form a transient peptide-DNA imino (Schiff base) intermediate, which may be isolated as a stable covalent species under reducing conditions. In the current study, we use this methodology to demonstrate that peptide-catalyzed beta-elimination proceeds via the formation of two Schiff base intermediates, one of which was covalently trapped prior to strand incision and the other following strand incision. N-Terminal acetylation of reactive peptides significantly inhibited formation of a trapped Schiff base complex; thus, we demonstrate for the first time that the preferred reactive nucleophile for peptides catalyzing strand incision is the N-terminal alpha-amino group, not an epsilon-amino group located on a lysine residue as previously postulated. Trapping reactions in which the central tryptophan residue was changed to alanine did not have a significant impact on the efficiency of Schiff base formation, indicating that the presence of an aromatic residue is dispensable for the step prior to peptide-catalyzed beta-elimination. Interestingly, the methodology presented here affords a convenient means for covalently attaching an array of peptides onto AP site-containing DNA in a site-specific fashion. We suggest that the generation of such DNA-peptide cross-links may provide utility in studying the repair of biologically significant DNA-protein cross-link damage as DNA-peptide complexes may mimic intermediate structures along a repair pathway for DNA-protein cross-links.

Redox effector factor-1 regulates the activity of thyroid transcription factor 1 by controlling the redox state of the N transcriptional activation domain.

Thyroid transcription factor 1 (TTF-1) is a homeodomain-containing transcriptional regulator responsible for the activation of thyroid- and lung-specific genes. It has been demonstrated that its DNA binding activity is redox-regulated in vitro through the formation of dimers and oligomeric species. In this paper, we demonstrate that the redox regulation mainly involves a Cys residue (Cys(87)), which resides out of the DNA binding domain, belonging to the N-transactivation domain. In fact, the oxidized form of a truncated TTF-1 (containing the N-transactivation domain and the DNA-binding domain, here called TTF-1N-HD) looses specific DNA binding activity. Since most of the oxidized TTF-1N-HD is in a monomeric form, these data indicate that the redox state of Cys(87) may control the DNA-binding function of the homeodomain, suggesting that Cys(87) could play an important role in determining the correct folding of the homeodomain. By using gel retardation and transient transfection assays, we demonstrate that the redox effector factor-1 (Ref-1) mediates the redox effects on TTF-1N-HD binding and that it is able to modulate the TTF-1 transcriptional activity. Glutathione S-transferase pull-down experiments demonstrate the occurrence of interaction between Ref-1 and TTF-1N-HD. Having previously demonstrated that Ref-1 is able to modulate the transcriptional activity of another thyroid-specific transcription factor (Pax-8), our data suggest that Ref-1 plays a central role in the regulation of thyroid cells.

Structure and function of threonine synthase from yeast.

Threonine synthase catalyzes the final step of threonine biosynthesis, the pyridoxal 5'-phosphate (PLP)-dependent conversion of O-phosphohomoserine into threonine and inorganic phosphate. Threonine is an essential nutrient for mammals, and its biosynthetic machinery is restricted to bacteria, plants, and fungi; therefore, threonine synthase represents an interesting pharmaceutical target. The crystal structure of threonine synthase from Saccharomyces cerevisiae has been solved at 2.7 A resolution using multiwavelength anomalous diffraction. The structure reveals a monomer as active unit, which is subdivided into three distinct domains: a small N-terminal domain, a PLP-binding domain that covalently anchors the cofactor and a so-called large domain, which contains the main of the protein body. All three domains show the typical open alpha/beta architecture. The cofactor is bound at the interface of all three domains, buried deeply within a wide canyon that penetrates the whole molecule. Based on structural alignments with related enzymes, an enzyme-substrate complex was modeled into the active site of yeast threonine synthase, which revealed essentials for substrate binding and catalysis. Furthermore, the comparison with related enzymes of the beta-family of PLP-dependent enzymes indicated structural determinants of the oligomeric state and thus rationalized for the first time how a PLP enzyme acts in monomeric form.

Molecular mechanism of PCNA-dependent base excision repair.

In higher eukaryotes, base excision repair can proceed by two alternative pathways: a DNA polymerase beta-dependent pathway and a proliferating cell nuclear antigen (PCNA)-dependent pathway. Recently, we have reconstituted the PCNA-dependent AP site repair reaction with six purified human proteins: AP endonuclease, replication factor C (RFC), PCNA, flap endonuclease 1 (FEN1), DNA polymerase delta (pol delta), and DNA ligase I. In this reconstituted system, the number of nucleotides replaced during the repair reaction (patch size) was predominantly two nucleotides. PCNA can directly interact with RFC, pol delta, FEN1 and DNA ligase I. These interactions are partly through a consensus motif, QXX(I/L/M)XX(F/H)(F/Y), found in each of the four proteins. PCNA functions as a molecular adaptor for recruiting these factors to the site of DNA repair. Two DNA-N-glycosylases among those so far cloned from human, UNG2 and MYH, are found to have the same PCNA-binding motif. Major substrates of these enzymes, a uracil opposite an adenine for UNG2 and an adenine opposite an 8-oxoguanine for MYH, are formed during DNA replication. Therefore, UNG2 and MYH may serve for replication-coupled base excision repair through the direct interaction with PCNA in the replication machinery.