ABSTRACT
BACKGROUND: The DNA replication checkpoint ensures that mitosis is not initiated before DNA synthesis is completed. Recent studies using Xenopus extracts have demonstrated that activation of the replication checkpoint and phosphorylation of the Chk1 kinase are dependent on RNA primer synthesis by DNA polymerase alpha, and it has been suggested that the ATR kinase-so-called because it is related to the product of the gene that is mutated in ataxia telangiectasia (ATM) and to Rad3 kinase-may be an upstream component of this response. It has been difficult to test this hypothesis as an ATR-deficient system suitable for biochemical studies has not been available. RESULTS: We have cloned the Xenopus laevis homolog of ATR (XATR) and studied the function of the protein in Xenopus egg extracts. Using a chromatin-binding assay, we found that ATR associates with chromatin after initiation of replication, dissociates from chromatin upon completion of replication, and accumulates in the presence of aphidicolin, an inhibitor of DNA replication. Its association with chromatin was inhibited by treatment with actinomycin D, an inhibitor of RNA primase. There was an early rise in the activity of Cdc2-cyclin B in egg extracts depleted of ATR both in the presence or absence of aphidicolin. In addition, the premature mitosis observed upon depletion of ATR was accompanied by the loss of Chk1 phosphorylation. CONCLUSIONS: ATR is a replication-dependent chromatin-binding protein, and its association with chromatin is dependent on RNA synthesis by DNA polymerase alpha. Depletion of ATR leads to premature mitosis in the presence and absence of aphidicolin, indicating that ATR is required for the DNA replication checkpoint.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatin/metabolism , DNA Replication , Genes, cdc/physiology , Protein Serine-Threonine Kinases , Xenopus Proteins , Amino Acid Sequence , Animals , Aphidicolin/pharmacology , Ataxia Telangiectasia Mutated Proteins , Blotting, Western , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Nucleus/metabolism , Checkpoint Kinase 1 , Cloning, Molecular , DNA Replication/drug effects , Dactinomycin/pharmacology , Enzyme Inhibitors/pharmacology , Humans , Male , Molecular Sequence Data , Oocytes/physiology , Protein Kinases/metabolism , Protein Synthesis Inhibitors/pharmacology , Recombinant Proteins/metabolism , Sequence Analysis, DNA , Spermatozoa/physiology , Xenopus laevisABSTRACT
With emerging drug resistance in Plasmodium falciparum, novel antifolates effective against pyrimethamine-resistant and cycloguanil-resistant dihydrofolate reductase (DHFR) are in demand. Based on structural similarity to cycloguanil, it has been proposed that WR99210, and its metabolic precursor PS-15, exerts selective antimalarial activity by binding tightly to both drug-sensitive and drug-resistant DHFR. In the present study, Linweaver-Burk plots and Ackermann-Potter plots reveal that both forms of malarial DHFR bind WR99210 at subnanomolar concentrations. It is not necessary to invoke an alternate target for WR99210 in P. falciparum. The present studies confirm that malarial DHFRs offer potential binding interactions in the folate-binding pocket distinct from those exploited by pyrimethamine and cycloguanil. These kinetic studies also provide a useful framework for the design and interpretation of future structural studies on drug-resistant DHFR from P. falciparum.
Subject(s)
Antimalarials/pharmacology , Folic Acid Antagonists/pharmacology , Plasmodium falciparum/enzymology , Pyrimethamine/pharmacology , Tetrahydrofolate Dehydrogenase/drug effects , Triazines/pharmacology , Animals , Dose-Response Relationship, Drug , Drug Resistance , KineticsSubject(s)
Antimalarials/pharmacology , Gene Expression Regulation, Enzymologic , Plasmodium falciparum/genetics , Pyrimethamine/pharmacology , Tetrahydrofolate Dehydrogenase/genetics , Animals , Cloning, Molecular , DNA Primers/chemistry , DNA, Protozoan/chemistry , DNA, Protozoan/genetics , Drug Resistance/genetics , Genetic Vectors , Plasmids , Plasmodium falciparum/drug effects , Plasmodium falciparum/enzymology , Polymerase Chain Reaction , Tetrahydrofolate Dehydrogenase/biosynthesisABSTRACT
Consistent with a proposed mechanism for the potent antimalarial activity of 5-fluoroorotate, 5-fluoro-2'-deoxyuridylate inhibited Plasmodium falciparum thymidylate synthase with a Ki of 2 nM. Steady-state kinetics revealed no significant differences between malarial and mammalian thymidylate synthases. Thus, additional biochemical parameters must underlie the selective antimalarial activity of 5-fluoroorotate. A polyglutamylated folate analog, D1694-(glu)4, was also a potent inhibitor of malarial thymidylate synthase (Kis = 1.5 nM).
