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Triaza-coumarin (TA-C) is a Mycobacterium tuberculosis (Mtb) dihydrofolate reductase (DHFR) inhibitor with an IC 50 (half maximal inhibitory concentration) of ∼1 µM against the enzyme. Despite this moderate target inhibition, TA-C shows exquisite antimycobacterial activity (MIC 50 , concentration inhibiting growth by 50% = 10 to 20 nM). Here, we investigated the mechanism underlying this potency disconnect. To confirm that TA-C targets DHFR and investigate its unusual potency pattern, we focused on resistance mechanisms. In Mtb, resistance to DHFR inhibitors is frequently associated with mutations in thymidylate synthase thyA , which sensitizes Mtb to DHFR inhibition, rather than in DHFR itself. We observed thyA mutations, consistent with TA-C interfering with the folate pathway. A second resistance mechanism involved biosynthesis of the redox coenzyme F 420 Thus, we hypothesized that TA-C may be metabolized by Mtb F 420 -dependent oxidoreductases (FDORs). By chemically blocking the putative site of FDOR-mediated reduction in TA-C, we reproduced the F 420 -dependent resistance phenotype, suggesting that F 420 H 2 -dependent reduction is required for TA-C to exert its potent antibacterial activity. Indeed, chemically synthesized TA-C-Acid, the putative product of TA-C reduction, displayed a 100-fold lower IC 50 against DHFR. Screening seven recombinant Mtb FDORs revealed that at least two of these enzymes reduce TA-C. This redundancy in activation explains why no mutations in the activating enzymes were identified in the resistance screen. Analysis of the reaction products confirmed that FDORs reduce TA-C at the predicted site, yielding TA-C-Acid. This work demonstrates that intrabacterial metabolism converts TA-C, a moderately active "prodrug," into a 100-fold-more-potent DHFR inhibitor, thus explaining the disconnect between enzymatic and whole-cell activity., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Many large proteins suffer from slow or inefficient folding in vitro. It has long been known that this problem can be alleviated in vivo if proteins start folding cotranslationally. However, the molecular mechanisms underlying this improvement have not been well established. To address this question, we use an all-atom simulation-based algorithm to compute the folding properties of various large protein domains as a function of nascent chain length. We find that for certain proteins, there exists a narrow window of lengths that confers both thermodynamic stability and fast folding kinetics. Beyond these lengths, folding is drastically slowed by nonnative interactions involving C-terminal residues. Thus, cotranslational folding is predicted to be beneficial because it allows proteins to take advantage of this optimal window of lengths and thus avoid kinetic traps. Interestingly, many of these proteins' sequences contain conserved rare codons that may slow down synthesis at this optimal window, suggesting that synthesis rates may be evolutionarily tuned to optimize folding. Using kinetic modeling, we show that under certain conditions, such a slowdown indeed improves cotranslational folding efficiency by giving these nascent chains more time to fold. In contrast, other proteins are predicted not to benefit from cotranslational folding due to a lack of significant nonnative interactions, and indeed these proteins' sequences lack conserved C-terminal rare codons. Together, these results shed light on the factors that promote proper protein folding in the cell and how biomolecular self-assembly may be optimized evolutionarily., Competing Interests: The authors declare no competing interest.

Fitness landscapes of drug resistance constitute powerful tools to elucidate mutational pathways of antibiotic escape. Here, we developed a predictive biophysics-based fitness landscape of trimethoprim (TMP) resistance for Escherichia coli dihydrofolate reductase (DHFR). We investigated the activity, binding, folding stability, and intracellular abundance for a complete set of combinatorial DHFR mutants made out of three key resistance mutations and extended this analysis to DHFR originated from Chlamydia muridarum and Listeria grayi We found that the acquisition of TMP resistance via decreased drug affinity is limited by a trade-off in catalytic efficiency. Protein stability is concurrently affected by the resistant mutants, which precludes a precise description of fitness from a single molecular trait. Application of the kinetic flux theory provided an accurate model to predict resistance phenotypes (IC50) quantitatively from a unique combination of the in vitro protein molecular properties. Further, we found that a controlled modulation of the GroEL/ES chaperonins and Lon protease levels affects the intracellular steady-state concentration of DHFR in a mutation-specific manner, whereas IC50 is changed proportionally, as indeed predicted by the model. This unveils a molecular rationale for the pleiotropic role of the protein quality control machinery on the evolution of antibiotic resistance, which, as we illustrate here, may drastically confound the evolutionary outcome. These results provide a comprehensive quantitative genotype-phenotype map for the essential enzyme that serves as an important target of antibiotic and anticancer therapies.

How do the folding mechanisms of multidomain proteins depend on protein topology? We addressed this question by developing an Ising-like structure-based model and applying it for the analysis of free-energy landscapes and folding kinetics of an example protein, Escherichia coli dihydrofolate reductase (DHFR). DHFR has two domains, one comprising discontinuous N- and C-terminal parts and the other comprising a continuous middle part of the chain. The simulated folding pathway of DHFR is a sequential process during which the continuous domain folds first, followed by the discontinuous domain, thereby avoiding the rapid decrease in conformation entropy caused by the association of the N- and C-terminal parts during the early phase of folding. Our simulated results consistently explain the observed experimental data on folding kinetics and predict an off-pathway structural fluctuation at equilibrium. For a circular permutant for which the topological complexity of wild-type DHFR is resolved, the balance between energy and entropy is modulated, resulting in the coexistence of the two folding pathways. This coexistence of pathways should account for the experimentally observed complex folding behavior of the circular permutant.

Cockayne syndrome type B ATPase (CSB) belongs to the SwItch/Sucrose nonfermentable family. Its mutations are linked to Cockayne syndrome phenotypes and classically are thought to be caused by defects in transcription-coupled repair, a subtype of DNA repair. Here we show that after UV-C irradiation, immediate early genes such as activating transcription factor 3 (ATF3) are overexpressed. Although the ATF3 target genes, including dihydrofolate reductase (DHFR), were unable to recover RNA synthesis in CSB-deficient cells, transcription was restored rapidly in normal cells. There the synthesis of DHFR mRNA restarts on the arrival of RNA polymerase II and CSB and the subsequent release of ATF3 from its cAMP response element/ATF target site. In CSB-deficient cells ATF3 remains bound to the promoter, thereby preventing the arrival of polymerase II and the restart of transcription. Silencing of ATF3, as well as stable introduction of wild-type CSB, restores RNA synthesis in UV-irradiated CSB cells, suggesting that, in addition to its role in DNA repair, CSB activity likely is involved in the reversal of inhibitory properties on a gene-promoter region. We present strong experimental data supporting our view that the transcriptional defects observed in UV-irradiated CSB cells are largely the result of a permanent transcriptional repression of a certain set of genes in addition to some defect in DNA repair.

With the rapidly growing wealth of genomic data, experimental inquiries on the functional significance of important divergence sites in protein evolution are becoming more accessible. Here we trace the evolution of dihydrofolate reductase (DHFR) and identify multiple key divergence sites among 233 species between humans and bacteria. We connect these sites, experimentally and computationally, to changes in the enzyme's binding properties and catalytic efficiency. One of the identified evolutionarily important sites is the N23PP modification (∼mid-Devonian, 415-385 Mya), which alters the conformational states of the active site loop in Escherichia coli dihydrofolate reductase and negatively impacts catalysis. This enzyme activity was restored with the inclusion of an evolutionarily significant lid domain (G51PEKN in E. coli enzyme; ∼2.4 Gya). Guided by this evolutionary genomic analysis, we generated a human-like E. coli dihydrofolate reductase variant through three simple mutations despite only 26% sequence identity between native human and E. coli DHFRs. Molecular dynamics simulations indicate that the overall conformational motions of the protein within a common scaffold are retained throughout evolution, although subtle changes to the equilibrium conformational sampling altered the free energy barrier of the enzymatic reaction in some cases. The data presented here provide a glimpse into the evolutionary trajectory of functional DHFR through its protein sequence space that lead to the diverged binding and catalytic properties of the E. coli and human enzymes.

Plasmodium falciparum causes the deadliest form of human malaria. Its virulence is attributed to its ability to modify the infected RBC and to evade human immune attack through antigenic variation. Antigenic variation is achieved through tight regulation of antigenic switches between variable surface antigens named "P. falciparum erythrocyte membrane protein-1" encoded by the var multicopy gene family. Individual parasites express only a single var gene at a time, maintaining the remaining var genes in a transcriptionally silent state. Strict pairing between var gene promoters and a second promoter within an intron found in each var gene is required for silencing and counting of var genes by the mechanism that controls mutually exclusive expression. We have identified and characterized insulator-like DNA elements that are required for pairing var promoters and introns and thus are essential for regulating silencing and mutually exclusive expression. These elements, found in the regulatory regions of each var gene, are bound by distinct nuclear protein complexes. Any alteration in the specific, paired structure of these elements by either deletion or insertion of additional elements results in an unregulated var gene. We propose a model by which silencing and mutually exclusive expression of var genes is regulated by the precise arrangement of insulator-like DNA pairing elements.

Toxoplasma gondii persistently infects over two billion people worldwide. It can cause substantial morbidity and mortality. Existing treatments have associated toxicities and hypersensitivity and do not eliminate encysted bradyzoites that recrudesce. New, improved medicines are needed. Transductive peptides carry small molecule cargos across multiple membranes to enter intracellular tachyzoites and encysted bradyzoites. They also carry cargos into retina when applied topically to eyes, and cross blood brain barrier when administered intravenously. Phosphorodiamidate morpholino oligomers (PMO) inhibit gene expression in a sequence-specific manner. Herein, effect of transductive peptide conjugated PMO (PPMO) on tachyzoite protein expression and replication in vitro and in vivo was studied. Initially, sequence-specific PPMO successfully reduced transfected T. gondii's fluorescence and luminescence. PPMO directed against T. gondii's dihydrofolate reductase (DHFR), an enzyme necessary for folate synthesis, limited tachyzoite replication. Rescue with exogenous folate demonstrated DHFR PPMO's specificity. PPMO directed against enoyl-ACP reductase (ENR), an enzyme of type II fatty acid synthesis that is structurally distinct in T. gondii from ENR in mammalian cells was investigated. PPMO directed against plant-like Apetela 2 (AP2) domain transcription factor XI-3 (AP2XI-3), not present in human cells, was characterized. ENR and AP2XI-3 PPMO each restricted intracellular parasite replication validating these molecular targets in tachyzoites. DHFR-specific PPMO administered to infected mice diminished parasite burden. Thus, these antisense oligomers are a versatile approach to validate T. gondii molecular targets, reduce essential T. gondii proteins in vitro and in vivo, and have potential for development as curative medicines.

Mutations create the genetic diversity on which selective pressures can act, yet also create structural instability in proteins. How, then, is it possible for organisms to ameliorate mutation-induced perturbations of protein stability while maintaining biological fitness and gaining a selective advantage? Here we used site-specific chromosomal mutagenesis to introduce a selected set of mostly destabilizing mutations into folA--an essential chromosomal gene of Escherichia coli encoding dihydrofolate reductase (DHFR)--to determine how changes in protein stability, activity, and abundance affect fitness. In total, 27 E. coli strains carrying mutant DHFR were created. We found no significant correlation between protein stability and its catalytic activity nor between catalytic activity and fitness in a limited range of variation of catalytic activity observed in mutants. The stability of these mutants is strongly correlated with their intracellular abundance, suggesting that protein homeostatic machinery plays an active role in maintaining intracellular concentrations of proteins. Fitness also shows a significant correlation with intracellular abundance of soluble DHFR in cells growing at 30 °C. At 42 °C, the picture was mixed, yet remarkable: A few strains carrying mutant DHFR proteins aggregated, rendering them nonviable, but, intriguingly, the majority exhibited fitness higher than wild type. We found that mutational destabilization of DHFR proteins in E. coli is counterbalanced at 42 °C by their soluble oligomerization, thereby restoring structural stability and protecting against aggregation.

Surveillance for drug-resistant parasites in human blood is a major effort in malaria control. Here we report contrasting antifolate resistance polymorphisms in Plasmodium falciparum when parasites in human blood were compared with parasites in Anopheles vector mosquitoes from sleeping huts in rural Zambia. DNA encoding P. falciparum dihydrofolate reductase (EC 1.5.1.3) was amplified by PCR with allele-specific restriction enzyme digestions. Markedly prevalent pyrimethamine-resistant mutants were evident in human P. falciparum infections--S108N (>90%), with N51I, C59R, and 108N+51I+59R triple mutants (30-80%). This resistance level may be from selection pressure due to decades of sulfadoxine/pyrimethamine use in the region. In contrast, cycloguanil-resistant mutants were detected in very low frequency in parasites from human blood samples-S108T (13%), with A16V and 108T+16V double mutants (∼4%). Surprisingly, pyrimethamine-resistant mutants were of very low prevalence (2-12%) in the midguts of Anopheles arabiensis vector mosquitoes, but cycloguanil-resistant mutants were highly prevalent-S108T (90%), with A16V and the 108T+16V double mutant (49-57%). Structural analysis of the dihydrofolate reductase by in silico modeling revealed a key difference in the enzyme within the NADPH binding pocket, predicting the S108N enzyme to have reduced stability but the S108T enzyme to have increased stability. We conclude that P. falciparum can bear highly host-specific drug-resistant polymorphisms, most likely reflecting different selective pressures found in humans and mosquitoes. Thus, it may be useful to sample both human and mosquito vector infections to accurately ascertain the epidemiological status of drug-resistant alleles.

Human dihydrofolate reductase (DHFR) was previously thought to be the only enzyme capable of the reduction of dihydrofolate to tetrahydrofolate; an essential reaction necessary to ensure a continuous supply of biologically active folate. DHFR has been studied extensively from a number of perspectives because of its role in health and disease. Although the presence of a number of intronless DHFR pseudogenes has been known since the 1980s, it was assumed that none of these were expressed or functional. We show that humans do have a second dihydrofolate reductase enzyme encoded by the former pseudogene DHFRP4, located on chromosome 3. We demonstrate that the DHFRP4, or dihydrofolate reductase-like 1 (DHFRL1), gene is expressed and shares some commonalities with DHFR. Recombinant DHFRL1 can complement a DHFR-negative phenotype in bacterial and mammalian cells but has a lower specific activity than DHFR. The K(m) for NADPH is similar for both enzymes but DHFRL1 has a higher K(m) for dihydrofolate when compared to DHFR. The need for a second reductase with lowered affinity for its substrate may fulfill a specific cellular requirement. The localization of DHFRL1 to the mitochondria, as demonstrated by confocal microscopy, indicates that mitochondrial dihydrofolate reductase activity may be optimal with a lowered affinity for dihydrofolate. We also found that DHFRL1 is capable of the same translational autoregulation as DHFR by binding to its own mRNA; with each enzyme also capable of replacing the other. The identification of DHFRL1 will have implications for previous research involving DHFR.

The de novo and salvage dTTP pathways are essential for maintaining cellular dTTP pools to ensure the faithful replication of both mitochondrial and nuclear DNA. Disregulation of dTTP pools results in mitochondrial dysfunction and nuclear genome instability due to an increase in uracil misincorporation. In this study, we identified a de novo dTMP synthesis pathway in mammalian mitochondria. Mitochondria purified from wild-type Chinese hamster ovary (CHO) cells and HepG2 cells converted dUMP to dTMP in the presence of NADPH and serine, through the activities of mitochondrial serine hydroxymethyltransferase (SHMT2), thymidylate synthase (TYMS), and a novel human mitochondrial dihydrofolate reductase (DHFR) previously thought to be a pseudogene known as dihydrofolate reductase-like protein 1 (DHFRL1). Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane, confirming the presence of this pathway in mitochondria. Knockdown of DHFRL1 using siRNA eliminated DHFR activity in mitochondria. DHFRL1 expression in CHO glyC, a previously uncharacterized mutant glycine auxotrophic cell line, rescued the glycine auxotrophy. De novo thymidylate synthesis activity was diminished in mitochondria isolated from glyA CHO cells that lack SHMT2 activity, as well as mitochondria isolated from wild-type CHO cells treated with methotrexate, a DHFR inhibitor. De novo thymidylate synthesis in mitochondria prevents uracil accumulation in mitochondrial DNA (mtDNA), as uracil levels in mtDNA isolated from glyA CHO cells was 40% higher than observed in mtDNA isolated from wild-type CHO cells. These data indicate that unlike other nucleotides, de novo dTMP synthesis occurs within mitochondria and is essential for mtDNA integrity.

Drug resistance resulting from mutations to the target is an unfortunate common phenomenon that limits the lifetime of many of the most successful drugs. In contrast to the investigation of mutations after clinical exposure, it would be powerful to be able to incorporate strategies early in the development process to predict and overcome the effects of possible resistance mutations. Here we present a unique prospective application of an ensemble-based protein design algorithm, K*, to predict potential resistance mutations in dihydrofolate reductase from Staphylococcus aureus using positive design to maintain catalytic function and negative design to interfere with binding of a lead inhibitor. Enzyme inhibition assays show that three of the four highly-ranked predicted mutants are active yet display lower affinity (18-, 9-, and 13-fold) for the inhibitor. A crystal structure of the top-ranked mutant enzyme validates the predicted conformations of the mutated residues and the structural basis of the loss of potency. The use of protein design algorithms to predict resistance mutations could be incorporated in a lead design strategy against any target that is susceptible to mutational resistance.

We report hydrogen deuterium exchange by mass spectrometry (HDX-MS) as a function of temperature in a thermophilic dihydrofolate reductase from Bacillus stearothermophilus (Bs-DHFR). Protein stability, probed with circular dichroism, established an accessible temperature range of 10 degrees C to 55 degrees C for the interrogation of HDX-MS. Although both the rate and extent of HDX are sensitive to temperature, the majority of peptides showed rapid kinetics of exchange, allowing us to focus on plateau values for the maximal extent of exchange at each temperature. Arrhenius plots of the ratio of hydrogens exchanged at 5 h normalized to the number of exchangeable hydrogens vs. 1/T provides an estimate for the apparent enthalpic change of local unfolding, DeltaH degrees (unf(avg)). Most regions in the enzyme show DeltaH degrees (unf(avg)) < or = 2.0 kcal/mol, close to the value of kT; by contrast, significantly elevated values for DeltaH degrees (unf(avg)) are observed in regions within the core of protein that contain the cofactor and substrate-binding sites. Our technique introduces a new strategy for probing the temperature dependence of local protein unfolding within native proteins. These findings are discussed in the context of the demonstrated role for nuclear tunneling in hydride transfer from NADPH to dihydrofolate, and relate the observed enthalpic changes to two classes of motion, preorganization and reorganization, that have been proposed to control the efficiency of hydrogenic wave function overlap. Our findings suggest that the enthalpic contribution to the heavy atom environmental reorganizations controlling the hydrogenic wave function overlap will be dominated by regions of the protein proximal to the bound cofactor and substrate.

The spread of high-level pyrimethamine resistance in Africa threatens to curtail the therapeutic lifetime of antifolate antimalarials. We studied the possible evolutionary pathways in the evolution of pyrimethamine resistance using an approach in which all possible mutational intermediates were created by site-directed mutagenesis and assayed for their level of drug resistance. The coding sequence for dihydrofolate reductase (DHFR) from the malaria parasite Plasmodium falciparum was mutagenized, and tests were carried out in Escherichia coli under conditions in which the endogenous bacterial enzyme was selectively inhibited. We studied 4 key amino acid replacements implicated in pyrimethamine resistance: N51I, C59R, S108N, and I164L. Using empirical estimates of the mutational spectrum in P. falciparum and probabilities of fixation based on the relative levels of resistance, we found that the predicted favored pathways of drug resistance are consistent with those reported in previous kinetic studies, as well as DHFR polymorphisms observed in natural populations. We found that 3 pathways account for nearly 90% of the simulated realizations of the evolution of pyrimethamine resistance. The most frequent pathway (S108N and then C59R, N51I, and I164L) accounts for more than half of the simulated realizations. Our results also suggest an explanation for why I164L is detected in Southeast Asia and South America, but not at significant frequencies in Africa.

Intermittent preventive treatment in pregnancy (IPTp) is used to prevent Plasmodium falciparum malaria. However, parasites resistant to the IPTp drug sulfadoxine-pyrimethamine (SP) have emerged worldwide, and infections with mixed resistant and susceptible parasites are exacerbated by pyrimethamine in mice. In a prospective delivery cohort in Muheza, Tanzania, we examined the effects of SP IPTp on parasite resistance alleles, parasite diversity, level of parasitemia, and inflammation in the placenta. IPTp use was associated with an increased fraction of parasites carrying the resistance allele at DHPS codon 581, an increase in the level of parasitemia, and more intense placental inflammation. The lowest mean level of parasite diversity and highest mean level of parasitemia occurred in women after recent IPTp use. These findings support a model of parasite release and facilitation, whereby the most highly resistant parasites out-compete less fit parasite populations and overgrow under drug pressure. Use of partially effective anti-malarial agents for IPTp may exacerbate malaria infections in the setting of widespread drug resistance.

Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural-but imperfect-DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR(-/-) cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2-3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR(-/-) cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production.

MicroRNAs are predicted to regulate approximately 30% of all human genes by targeting sequences in their 3' UTR. Polymorphisms in 3' UTR of several genes have been reported to affect gene expression, but the mechanism is not fully understood. Here, we demonstrate that 829C-->T, a naturally occurring SNP, near the miR-24 binding site in the 3' UTR of human dihydrofolate reductase (DHFR) affects DHFR expression by interfering with miR-24 function, resulting in DHFR overexpression and methotrexate resistance. miR-24 has a conserved binding site in DHFR 3' UTR. DHFR with WT and 3' UTR containing the 829C-->T mutation were expressed in DG44 cells that lack DHFR. Overexpression of miR-24 in cells with WT DHFR resulted in down-regulation of DHFR protein, whereas no effect on DHFR protein expression was observed in the mutant 3' UTR-expressing cells. Inhibition of endogenous miR-24 with a specific inhibitor led to up-regulation of DHFR in WT and not in mutant cells. Cells with the mutant 3' UTR had a 2-fold increase in DHFR mRNA half-life, expressed higher DHFR mRNA and DHFR protein, and were 4-fold more resistant to methotrexate as compared with WT cells. SNP-829C-->T, therefore, leads to a decrease in microRNA binding leading to overexpression of its target and results in resistance to methotrexate. We demonstrate that a naturally occurring miRSNP (a SNP located at or near a microRNA binding site in 3' UTR of the target gene or in a microRNA) is associated with enzyme overproduction and drug resistance.

One of the most intriguing questions in modern enzymology is whether enzyme dynamics evolved to enhance the catalyzed chemical transformation. In this study, dihydrofolate reductase, a small monomeric protein that catalyzes a single C-H-C transfer, is used as a model system to address this question. Experimental and computational studies have proposed a dynamic network that includes two residues remote from the active site (G121 and M42). The current study compares the nature of the H-transfer step of the WT enzyme, two single mutants, and their double mutant. The contribution of quantum mechanical tunneling and enzyme dynamics to the H-transfer step was examined by determining intrinsic kinetic isotope effects, their temperature dependence, and activation parameters. Different patterns of environmentally coupled tunneling were found for these four enzymes. The findings indicate that the naturally evolved WT dihydrofolate reductase requires no donor-acceptor distance fluctuations (no gating). Both single mutations affect the rearrangement of the system before tunneling, so some gating is required, but the overall nature of the environmentally coupled tunneling appears similar to that of the WT enzyme. The double mutation, on the other hand, seems to cause a major change in the nature of H transfer, leading to poor reorganization and substantial gating. These findings support the suggestion that these distal residues synergistically affect the H transfer at the active site of the enzyme. This observation is in accordance with the notion that these remote residues are part of a dynamic network that is coupled to the catalyzed chemistry.

Pyrimethamine (Pyr) targets dihydrofolate reductase of Plasmodium vivax (PvDHFR) as well as other malarial parasites, but its use as antimalarial is hampered by the widespread high resistance. Comparison of the crystal structures of PvDHFR from wild-type and the Pyr-resistant (SP21, Ser-58 --> Arg + Ser-117 --> Asn) strain as complexes with NADPH and Pyr or its analog lacking p-Cl (Pyr20) clearly shows that the steric conflict arising from the side chain of Asn-117 in the mutant enzyme, accompanied by the loss of binding to Ser-120, is mainly responsible for the reduction in binding of Pyr. Pyr20 still effectively inhibits both the wild-type and SP21 proteins, and the x-ray structures of these complexes show how Pyr20 fits into both active sites without steric strain. These structural insights suggest a general approach for developing new generations of antimalarial DHFR inhibitors that, by only occupying substrate space of the active site, would retain binding affinity with the mutant enzymes.

A comprehensive analysis of the network of coupled motions correlated to hydride transfer in dihydrofolate reductase is presented. Hybrid quantum/classical molecular dynamics simulations are combined with a rank correlation analysis method to extract thermally averaged properties that vary along the collective reaction coordinate according to a prescribed target model. Coupled motions correlated to hydride transfer are identified throughout the enzyme. Calculations for wild-type dihydrofolate reductase and a triple mutant, along with the associated single and double mutants, indicate that each enzyme system samples a unique distribution of coupled motions correlated to hydride transfer. These coupled motions provide an explanation for the experimentally measured nonadditivity effects in the hydride transfer rates for these mutants. This analysis illustrates that mutations distal to the active site can introduce nonlocal structural perturbations and significantly impact the catalytic rate by altering the conformational motions of the entire enzyme and the probability of sampling conformations conducive to the catalyzed reaction.

Reverse genetics in Plasmodium, the genus of parasites that cause malaria, still faces major limitations. Only red blood cell stages of this haploid parasite can be transfected. Consequently, the function of many essential genes in these and subsequent stages, including those encoding vaccine candidates, cannot be addressed genetically. Here, we establish conditional mutagenesis in Plasmodium by using site-specific recombination and the Flp/FRT system of yeast. Site-specific recombination is induced after cross-fertilization in the mosquito vector of two clones containing either the target sequence flanked by two FRT sites or the Flp recombinase. Parasites that have undergone recombination are recognized in the cross progeny through the expression of a fluorescence marker. This approach should permit to dissect the function of any essential gene of Plasmodium during the haploid phase of its life, i.e., during infection of salivary glands in the mosquito and infection of both the liver and red blood cells in the mammal.

This article introduces the computational procedure FamClash for analyzing incompatibilities in engineered protein hybrids by using protein family sequence data. All pairs of residue positions in the sequence alignment that conserve the property triplet of charge, volume, and hydrophobicity are first identified, and significant deviations are denoted as residue-residue clashes. This approach moves beyond earlier efforts aimed at solely classifying hybrids as functional or nonfunctional by correlating the rank ordering of these hybrids based on their activity levels. Experimental testing of this approach was performed in parallel to assess the predictive ability of FamClash. As a model system, single-crossover ITCHY (incremental truncation for the creation of hybrid enzymes) libraries were prepared from the Escherichia coli and Bacillus subtilis dihydrofolate reductases, and the activities of functional hybrids were determined. Comparisons of the predicted clash map as a function of crossover position revealed good agreement with activity data, reproducing the observed V shape and matching the location of a local peak in activity.

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate. The catalytic rate in this system has been found to be significantly affected by mutations far from the site of chemical activity in the enzyme [Rajagopalan, P. T. R, Lutz, S., and Benkovic, S. J. (2002) Biochemistry 41, 12618-12628]. On the basis of extensive computer simulations for wild-type DHFR from Escherichia coli and four mutants (G121S, G121V, M42F, and M42F/G121S), we show that key parameters for catalysis are changed. The parameters we study are relative populations of different conformations sampled and hydrogen bonds. We find that the mutations result in long-range structural perturbations, rationalizing the effects that the mutations have on the kinetics of the enzyme. Such perturbations also provide a rationalization for the reported nonadditivity effect for double mutations. We finally examine the role a structural perturbation will have on the hydride transfer step. On the basis of our new findings, we discuss the role of coupled motions between distant regions in the enzyme, which previously was reported by Radkiewicz and Brooks.

Centered in the Chinese hamster dihydrofolate reductase origin of replication is a prominent nuclear matrix attachment region (MAR). Indirect lines of evidence suggested that this MAR might be required for origin activation in early S phase. To test this possibility, we have deleted the MAR from a Chinese hamster ovary variant harboring a single copy of the dihydrofolate reductase locus. However, 2D gel replicon mapping shows that removal of the MAR has no significant effect either on the frequency or timing of initiation in this locus. Rather, fluorescence in situ hybridization studies on cells swollen under either neutral or alkaline conditions show that deletion of the MAR interferes with local separation of daughter chromatids. This surprising result provides direct genetic evidence that at least a subset of MARs performs an important biological function, possibly related to chromatid cohesion and separation.

The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s(-1) and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.

Plasmodium vivax is a major public health problem in Asia and South and Central America where it is most prevalent. Until very recently, the parasite has been effectively treated with chloroquine, but resistance to this drug has now been reported in several areas. Affordable alternative treatments for vivax malaria are urgently needed. Pyrimethamine-sulfadoxine is an inhibitor of dihydrofolate reductase (DHFR) that has been widely used to treat chloroquine-resistant Plasmodium falciparum malaria. DHFR inhibitors have not been considered for treatment of vivax malaria, because initial trials showed poor efficacy against P. vivax. P. vivax cannot be grown in culture; the reason for its resistance to DHFR inhibitors is unknown. We show that, like P. falciparum, point mutations in the dhfr gene can cause resistance to pyrimethamine in P. vivax. WR99210 is a novel inhibitor of DHFR, effective even against the most pyrimethamine-resistant P. falciparum strains. We have found that it is also an extremely effective inhibitor of the P. vivax DHFR, and mutations that confer high-level resistance to pyrimethamine render the P. vivax enzyme exquisitely sensitive to WR99210. These data suggest that pyrimethamine and WR99210 would exert opposing selective forces on the P. vivax population. If used in combination, these two drugs could greatly slow the selection of parasites resistant to both drugs. If that is the case, this novel class of DHFR inhibitors could provide effective and affordable treatment for chloroquine- and pyrimethamine-resistant vivax and falciparum malaria for many years to come.

Gene amplification is a common form of genomic instability in a wide variety of organisms and is often associated with tumor progression in mammals. One striking feature of many amplified genes is their organization as large inverted duplications (palindromes). Here, we describe a molecular mechanism for palindrome formation in mammalian cells that is also conserved in protists. We introduced a short (79 or 229 bp) inverted repeat into the genome of Chinese hamster ovary cells and showed that it promoted the formation of a large DNA palindrome after an adjacent DNA double-strand break. This finding suggests that short inverted repeats in the mammalian genome can have a critical role in the initiation of gene amplification. This specific mechanism may provide a novel target for cancer therapies.

Human cells exposed to antifolates show a rapid increase in the levels of the enzyme dihydrofolate reductase (DHFR). We hypothesized that this adaptive response mechanism can be used to elevate cellular levels of proteins fused to DHFR. In this study, mouse cells transfected to express a green fluorescent protein-DHFR fusion protein and subsequently exposed to the antifolate trimetrexate (TMTX) showed a specific and time-dependent increase in cellular levels of the fusion protein. Next, human HCT-8 and HCT-116 colon cancer cells retrovirally transduced to express a DHFR-herpes simplex virus 1 thymidine kinase (HSV1 TK) fusion protein and treated with the DHFR inhibitor TMTX exhibited increased levels of the DHFR-HSV1 TK fusion protein and an increase in ganciclovir sensitivity by 250-fold. The level of fusion protein in antifolate-treated human tumor cells was increased in response to a 24-h exposure of methotrexate, trimetrexate, as well as dihydrofolate. This effect depended on the antifolate concentration and was independent of the fusion-protein mRNA levels, consistent with this increase occurring at a translational level. In a xenograft model, nude rats bearing DHFR-HSV1 TK-transduced HCT-8 tumors and treated with TMTX showed, after 24 h, a 2- to 4-fold increase of fusion-protein levels in tumor tissue from treated animals compared with controls, as determined by Western blotting. The fusion-protein increase was imaged with positron-emission tomography, where a substantially enhanced signal of the transduced tumor was detected in animals after antifolate administration. Drug-mediated elevation of cellular DHFR-fused proteins is a very useful method to modulate gene expression in vivo for imaging as well as therapeutic purposes.

A network of coupled promoting motions in the enzyme dihydrofolate reductase is identified and characterized. The present identification is based on genomic analysis for sequence conservation, kinetic measurements of multiple mutations, and mixed quantum/classical molecular dynamics simulations of hydride transfer. The motions in this network span time scales of femtoseconds to milliseconds and are found on the exterior of the enzyme as well as in the active site. This type of network has broad implications for an expanded role of the protein fold in catalysis as well as ancillaries such as the engineering of altered protein function and the action of drugs distal to the active site.

Hypermutagenic PCR has been used to simulate pseudogene evolution of the Escherichia coli R67 dihydrofolate reductase gene. Each time the most divergent clone was used as template for another round of hypermutagenesis. After six rounds, with an average mutation rate of 0.05 per base per round, up to a 46% nucleic acid sequence variation was achieved. For a few clones the protein information content could be annihilated. As the intermediates were cloned and sequenced, it was possible to establish the real lineage and compute the true number of mutations. Not surprisingly the true number of forward and back mutations as well as variable sites exceeded those based on comparing any single intermediate to the initial sequence. However, the true number of forward and backward mutations, as well as the number of variable sites, increased linearly with sequence divergence from the original sequence, suggesting an empirical means to correct for branch lengths.

DNA sequence amplification is one of the most frequent manifestations of genomic instability in human tumors. We have shown previously that amplification of the dihydrofolate reductase (DHFR) gene in Chinese hamster cells is initiated by chromosome breaks, followed by bridge-breakage-fusion cycles that generate large intrachromosomal repeats; these are ultimately trimmed by an unknown process to smaller, more homogenous units manifested as homogenously staining chromosome regions (HSRs). However, in most human tumor cells, amplified DNA sequences are borne on unstable, extrachromosomal double minutes (DMs), which suggests the operation of a different amplification mechanism. In this study, we have isolated a large number of independent methotrexate-resistant human cell lines, all of which contained DHFR-bearing DMs. Surprisingly, all but one of these also had suffered partial or complete loss of one of the parental DHFR-bearing chromosomes. Cells in a few populations displayed what could be transient intermediates in the amplification process, including an initial HSR, its subsequent breakage, the appearance of DHFR-containing fragments, and, finally, DMs. Our studies suggest that HSRs and DMs both are initiated by chromosome breaks, but that cell types differ in how the extra sequences ultimately are processed and/or maintained.

Current cell-free protein synthesis systems can synthesize proteins with high speed and accuracy, but produce only a low yield because of their instability over time. Here we describe the preparation of a highly efficient but also robust cell-free system from wheat embryos. We first investigated the source of the instability of existing systems in light of endogenous ribosome-inactivating proteins and found that ribosome inactivation by tritin occurs already during extract preparation and continues during incubation for protein synthesis. Therefore, we prepared our system from extensively washed embryos that are devoid of contamination by endosperm, the source of tritin and possibly other inhibitors. In a batch system, we observed continuous translation for 4 h, and sucrose density gradient analysis showed formation of large polysomes, indicating high protein synthesis activity. When the reaction was performed in a dialysis bag, enabling the continuous supply of substrates together with the continuous removal of small byproducts, translation proceeded for >60 h, yielding 1-4 mg of enzymatically active proteins, and 0.6 mg of a 126-kDa tobacco mosaic virus protein, per milliliter of reaction volume. Our results demonstrate that plants contain endogenous inhibitors of translation and that after their elimination the translational apparatus is very stable. This contrasts with the common belief that cell-free translation systems are inherently unstable, even fragile. Our method is useful for the preparation of large amounts of active protein as well as for the study of protein synthesis itself.

Combinatorial libraries of synthetic and natural products are an important source of molecular information for the interrogation of biological targets. Methods for the intracellular production of libraries of small, stable molecules would be a valuable addition to existing library technologies by combining the discovery potential inherent in small molecules with the large library sizes that can be realized by intracellular methods. We have explored the use of split inteins (internal proteins) for the intracellular catalysis of peptide backbone cyclization as a method for generating proteins and small peptides that are stabilized against cellular catabolism. The DnaE split intein from Synechocystis sp. PCC6803 was used to cyclize the Escherichia coli enzyme dihydrofolate reductase and to produce the cyclic, eight-amino acid tyrosinase inhibitor pseudostellarin F in bacteria. Cyclic dihydrofolate reductase displayed improved in vitro thermostability, and pseudostellarin F production was readily apparent in vivo through its inhibition of melanin production catalyzed by recombinant Streptomyces antibioticus tyrosinase. The ability to generate and screen for backbone cyclic products in vivo is an important milestone toward the goal of generating intracellular cyclic peptide and protein libraries.

Two strategies are described for detecting constitutive or induced protein-protein interactions in intact mammalian cells; these strategies are based on oligomerization domain-assisted complementation of rationally designed fragments of the murine enzyme dihydrofolate reductase (DHFR; EC 1.5.1.3). We describe a dominant clonal-selection assay of stably transfected cells expressing partner proteins FKBP (FK506 binding protein) and FRAP (FKBP-rapamycin binding protein) fused to DHFR fragments and show a rapamycin dose-dependent survival of clones that requires approximately 25 molecules of reconstituted DHFR per cell. A fluorescence assay also is described, based on stoichiometric binding of fluorescein-methotrexate to reconstituted DHFR in vivo. Formation of the FKBP-rapamycin-FRAP complex is detected in stably and transiently transfected cells. Quantitative rapamycin dose-dependence of this complex is shown to be consistent with in vitro binding and distinguishable from a known constitutive interaction of FKBP and FRAP. We also show that this strategy can be applied to study membrane protein receptors, demonstrating dose-dependent activation of the erythropoietin receptor by ligands. The combination of these clonal-selection and fluorescence assays in intact mammalian cells makes possible selection by simple survival, flow cytometry, or both. High-throughput drug screening and quantitative analysis of induction or disruption of protein-protein interactions are also made possible.

Reassembly of enzymes from peptide fragments has been used as a strategy for understanding the evolution, folding, and role of individual subdomains in catalysis and regulation of activity. We demonstrate an oligomerization-assisted enzyme reassembly strategy whereby fragments are covalently linked to independently folding and interacting domains whose interactions serve to promote efficient refolding and complementation of fragments, forming active enzyme. We show that active murine dihydrofolate reductase (E.C. 1.5.1.3) can be reassembled from complementary N- and C-terminal fragments when fused to homodimerizing GCN4 leucine zipper-forming sequences as well as heterodimerizing protein partners. Reassembly is detected by an in vivo selection assay in Escherichia coli and in vitro. The effects of mutations that disrupt fragment affinity or enzyme activity were assessed. The steady-state kinetic parameters for the reassembled mutant (Phe-31 --> Ser) were determined; they are not significantly different from the full-length mutant. The strategy described here provides a general approach for protein dissection and domain swapping studies, with the capacity both for rapid in vivo screening as well as in vitro characterization. Further, the strategy suggests a simple in vivo enzyme-based detection system for protein-protein interactions, which we illustrate with two examples: ras-GTPase and raf-ras-binding domain and FK506-binding protein-rapamycin complexed with the target of rapamycin TOR2.

We tested the ability of recombinant hMutSalpha (hMSH2/hMSH6) and hMutSbeta (hMSH2/hMSH3) heterodimers to complement the mismatch repair defect of HEC59, a human cancer cell line whose extracts lack all three MutS homologues. Although repair of both base/base mispairs and insertion-deletion loops was restored by hMutSalpha, only the latter substrates were addressed in extracts supplemented with hMutSbeta. hMutSalpha was also able to complement a defect in the repair of base/base mispairs in CHO R and HL60R cell extracts. In these cells, methotrexate-induced amplification of the dihydrofolate reductase (DHFR) locus, which also contains the MSH3 gene, led to an overexpression of MSH3 and thus to a dramatic change in the relative levels of MutSalpha and MutSbeta. As a rule, MSH2 is primarily complexed with MSH6. MutSalpha is thus relatively abundant in mammalian cell extracts, whereas MutSbeta levels are generally low. In contrast, in cells that overexpress MSH3, the available MSH2 protein is sequestered predominantly into MutSbeta. This leads to degradation of the partnerless MSH6 and depletion of MutSalpha. CHO R and HL60R cells therefore lack correction of base/base mispairs, whereas loop repair is maintained by MutSbeta. Consequently, frameshift mutations in CHO R are rare, whereas transitions and transversions are acquired at a rate two orders of magnitude above background. Our data thus support and extend the findings of Drummond et al. [Drummond, J. T., Genschel, J., Wolf, E. & Modrich, P. (1997) Proc. Natl. Acad. Sci. USA 94, 10144-10149] and demonstrate that mismatch repair deficiency can arise not only through mutation or transcriptional silencing of a mismatch repair gene, but also as a result of imbalance in the relative amounts of the MSH3 and MSH6 proteins.

We have analyzed DNA sequences from world-wide geographic strains of Plasmodium falciparum and found a complete absence of synonymous DNA polymorphism at 10 gene loci. We hypothesize that all extant world populations of the parasite have recently derived (within several thousand years) from a single ancestral strain. The upper limit of the 95% confidence interval for the time when this most recent common ancestor lived is between 24,500 and 57,500 years ago (depending on different estimates of the nucleotide substitution rate); the actual time is likely to be much more recent. The recent origin of the P. falciparum populations could have resulted from either a demographic sweep (P. falciparum has only recently spread throughout the world from a small geographically confined population) or a selective sweep (one strain favored by natural selection has recently replaced all others). The selective sweep hypothesis requires that populations of P. falciparum be effectively clonal, despite the obligate sexual stage of the parasite life cycle. A demographic sweep that started several thousand years ago is consistent with worldwide climatic changes ensuing the last glaciation, increased anthropophilia of the mosquito vectors, and the spread of agriculture. P. falciparum may have rapidly spread from its African tropical origins to the tropical and subtropical regions of the world only within the last 6,000 years. The recent origin of the world-wide P. falciparum populations may account for its virulence, as the most malignant of human malarial parasites.

Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR-thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (DHFR) and MSH3 genes. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutSalpha (hMSH2. hMSH6) and hMutSbeta (hMSH2.hMSH3). In HL-60 extracts the hMutSalpha to hMutSbeta ratio is roughly 6:1, whereas in methotrexate-resistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of base-base mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (HPRT) locus. Purified hMutSalpha and hMutSbeta display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion-deletion heterology, but only hMutSalpha restores base-base mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.

Microgonotropen (MGT) DNA binding drugs, which consist of an A+T-selective DNA minor groove binding tripyrrole peptide and polyamine chains attached to a central pyrrole that extend drug contact into the DNA major groove, were found to be extraordinarily effective inhibitors of E2 factor 1 (E2F1) association with its DNA promoter element (5'-TTTCGCGCCAAA). The most active of these drugs, MGT-6a, was three orders of magnitude more effective than distamycin and inhibited complexes between E2F1 and the dihydrofolate reductase promoter by 50% at 0.00085 microM. A relationship was found between the measured equilibrium constants for binding of MGTs to the A+T region of d(GGCGA3T3GGC)/d(CCGCT3A3CCG) and their inhibition of complex formation between E2F1 and the DNA promoter element. A representative of the potent MGT inhibitors was significantly more active on inhibition of E2F1-DNA complex formation compared with disruption of a preexisting complex.

Single and multiple mutations at residues 16, 51, 59, 108, and 164 of Plasmodium falciparum dihydrofolate reductase (pfDHFR) have been linked to antifolate resistance in malaria. We prepared and characterized all seven of the pfDHFR mutants found in nature, as well as six mutants not observed in nature. Mutations involving residues 51, 59, 108, or 164 conferred cross resistance to both the antifolates pyrimethamine and cycloguanil, whereas mutation of residue 16 specifically conferred resistance to cycloguanil. The antifolate resistance of enzyme mutants found in nature correlated with in vivo antifolate resistance; however, mutants not found in nature were either poorly resistant or had insufficient catalytic activity to support DNA synthesis. Thus, specific combinations of multiple mutations at target residues were selected in nature to optimize resistance. Further, the resistance of multiple mutants was more than the sum of the component single mutations, indicating that residues were selected for their synergistic as well as intrinsic effects on resistance. Pathways inferred for the evolution of pyrimethamine-resistant mutants suggested that all multiple mutants emerged from stepwise selection of the single mutant, S108N. Thus, we propose that drugs targeted to both the wild-type pfDHFR and S108N mutant would have a low propensity for developing resistance, and hence could provide effective antimalarial agents.

The chaperonin GroEL binds nonnative proteins in its central channel through hydrophobic interactions and initiates productive folding in this space underneath bound co-chaperone, GroES, in the presence of ATP. The questions of where along the folding pathway a protein is recognized by GroEL, and how much structure is present in a bound substrate have remained subjects of discussion, with some experiments suggesting that bound forms are fully unfolded and others suggesting that bound species are partially structured. Here we have studied a substrate protein, human dihydrofolate reductase (DHFR), observing in stopped-flow fluorescence experiments that it can rapidly bind to GroEL at various stages of folding. We have also analyzed the structure of the GroEL-bound protein using hydrogen-deuterium exchange and NMR spectroscopy. The pattern and magnitude of amide proton protection indicate that the central parallel beta-sheet found in native DHFR is present in a moderately stable state in GroEL-bound DHFR. Considering that the strands are derived from distant parts of the primary structure, this suggests that a native-like global topology is also present. We conclude that significant native-like structure is present in protein-folding intermediates bound to GroEL.

In response to a moderate dose of radiation, asynchronous mammalian cell populations rapidly and transiently down-regulate the rate of DNA synthesis to approximately 50% of preirradiation values. We show here that only half of the reduction in overall replication rate can be accounted for by direct inhibition of initiation at origins in S-phase cells. The other half results from the operation of a newly defined cell cycle checkpoint that functions at the G1/S transition. This checkpoint senses damage incurred at any time during the last 2 hr of G1 and effectively prevents entry into the S period. The G1/S and S-phase checkpoints are both p53-independent and, unlike the p53-mediated G1 checkpoint, respond rapidly to radiation, suggesting that they may represent major damage-sensing mechanisms connecting the replication machinery with DNA repair pathways.

For use of ribozymes in vivo, it is desirable to select functional ribozymes in the cellular environment (in the presence of inhibitory factors and limited concentrations of mandatory Mg2+ ions, etc.). As a first step toward this goal, we developed a new screening system for detection in vivo of an active ribozyme from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker. In our DHFR expression vector, the sequence encoding either the active or the inactive ribozyme was connected to the DHFR gene. The plasmid was designed such that, when the ribozyme was active, the rate of production of DHFR was high enough to endow resistance to trimethoprim (TMP). We demonstrated that the active ribozyme did indeed cleave the primary transcript in vivo, whereas the inactive ribozyme had no cleavage activity. Cells that harbored the active-ribozyme-coding plasmid grew faster in the presence of a fixed concentration of TMP than the corresponding cells that harbored the inactive-ribozyme-coding plasmid. Consequently, when cells were transformed by a mixture that consisted of active- and inactive-ribozyme-coding plasmids at a ratio of 1:1, (i) mainly those cells that harbored active ribozymes survived in the presence of TMP and (ii) both active- and inactive-ribozyme-harboring cells grew at an identical rate in the absence of TMP, a demonstration of a positive selection system in vivo. If the background "noise" can be removed completely in the future, the selection system might usefully complement existing selection systems in vitro.

Nucleosomes, the basic structural elements of chromosomes, consist of 146 bp of DNA coiled around an octamer of histone proteins, and their presence can strongly influence gene expression. Considerations of the anisotropic flexibility of nucleotide triplets containing 3 cytosines or guanines suggested that a [5'(G/C)3 NN3']n motif might resist wrapping around a histone octamer. To test this, DNAs were constructed containing a 5'-CCGNN-3' pentanucleotide repeat with the Ns varied. Using in vitro nucleosome reconstitution and electron microscopy, a plasmid with 48 contiguous CCGNN repeats strongly excluded nucleosomes in the repeat region. Competitive reconstitution gel retardation experiments using DNA fragments containing 12, 24, or 48 CCGNN repeats showed that the propensity to exclude nucleosomes increased with the length of the repeat. Analysis showed that a 268-bp DNA containing a (CCGNN)48 block is 4.9 +/- 0.6-fold less efficient in nucleosome assembly than a similar length pUC19 fragment and approximately 78-fold less efficient than a similar length (CTG)n sequence, based on results from previous studies. Computer searches against the GenBank database for matches with a [(G/C)3NN]48 sequence revealed numerous examples that frequently were present in the control regions of "TATA-less" genes, including the human ETS-2 and human dihydrofolate reductase genes. In both cases the (G/C)3NN repeat, present in the promoter region, co-maps with loci previously shown to be nuclease hypersensitive sites.

Genetic studies of the protozoan parasite Plasmodium falciparum have been severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falciparum using a Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene, modified to confer resistance to pyrimethamine, as a selectable marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences. Transfected parasites generally maintained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid were obtained. Integration occurred by both homologous and nonhomologous recombination. In addition to the flanking sequence of the P. falciparum calmodulin gene, the 5' sequences of the P. falciparum and P. chabaudi dihydrofolate reductase-thymidylate synthase genes were also shown to be transcriptionally active in P. falciparum. The minimal 5' sequence that possessed significant transcriptional activity was determined for each gene and short sequences containing important transcriptional control elements were identified. These sequences will provide considerable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or modification of P. falciparum genes of interest.

To study the effect of apoptosis on gene amplification, we have constructed HeLa S3 cell lines in which the expression of bcl-2 (BCL2) can be controlled by tetracycline in the growth medium. Induction of Bcl-2 expression caused a temporary delay of apoptosis and resulted in roughly a 3-fold increase in the frequency of resistant colonies when cells were selected with trimetrexate. This resistance was due to amplification of the dihydrofolate reductase gene. Cells grown out of the pooled resistant colonies retained the same level of resistance to trimetrexate whether Bcl-2 was induced or repressed, consistent with the theory that Bcl-2 functions by facilitating gene amplification, rather than being the resistance mechanism per se. Pretreating cells with aphidicolin is another method to increase gene amplification frequency. When Bcl-2-expressing cells were pretreated with aphidicolin, the resulting increase in gene amplification frequency was approximately the product of the increases caused by aphidicolin pretreatment or Bcl-2 expression alone, indicating that Bcl-2 increases gene amplification through a mechanism independent of that of aphidicolin pretreatment. These results are consistent with the concept that gene amplification occurs at a higher frequency during drug-induced cell cycle perturbation. Bcl-2 evidently increases the number of selected amplified colonies by prolonging cell survival during the perturbation.

Plasmodium falciparum malaria parasites were transformed with plasmids containing P. falciparum or Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (dhfr-ts) coding sequences that confer resistance to pyrimethamine. Under pyrimethamine pressure, transformed parasites were obtained that maintained the transfected plasmids as unrearranged episomes for several weeks. These parasite populations were replaced after 2 to 3 months by parasites that had incorporated the transfected DNA into nuclear chromosomes. Depending upon the particular construct used for transformation, homologous integration was detected in the P. falciparum dhfr-ts locus (chromosome 4) or in hrp3 and hrp2 sequences that were used in the plasmid constructs as gene control regions (chromosomes 13 and 8, respectively). Transformation by homologous integration sets the stage for targeted gene alterations and knock-outs that will advance understanding of P. falciparum.