The replicase of all cells is thought to utilize two DNA polymerases for coordinated synthesis of leading and lagging strands. The DNA polymerases are held to DNA by circular sliding clamps. We demonstrate here that the E. coli DNA polymerase III holoenzyme assembles into a particle that contains three DNA polymerases. The three polymerases appear capable of simultaneous activity. Furthermore, the trimeric replicase is fully functional at a replication fork with helicase, primase, and sliding clamps; it produces slightly shorter Okazaki fragments than replisomes containing two DNA polymerases. We propose that two polymerases can function on the lagging strand and that the third DNA polymerase can act as a reserve enzyme to overcome certain types of obstacles to the replication fork.
Equilibrative transporters are potential drug targets, however most functional assays involve radioactive substrate uptake that is unsuitable for high-throughput screens (HTS). We developed a robust yeast-based growth assay that is potentially applicable to many equilibrative transporters. As proof of principle, we applied our approach to Equilibrative Nucleoside Transporter 1 of the malarial parasite Plasmodium falciparum (PfENT1). PfENT1 inhibitors might serve as novel antimalarial drugs since PfENT1-mediated purine import is essential for parasite proliferation. To identify PfENT1 inhibitors, we screened 64,560 compounds and identified 171 by their ability to rescue the growth of PfENT1-expressing fui1Δ yeast in the presence of a cytotoxic PfENT1 substrate, 5-fluorouridine (5-FUrd). In secondary assays, nine of the highest activity compounds inhibited PfENT1-dependent growth of a purine auxotrophic yeast strain with adenosine as the sole purine source (IC50 0.2–2 µM). These nine compounds completely blocked [3H]adenosine uptake into PfENT1-expressing yeast and erythrocyte-free trophozoite-stage parasites (IC50 5–50 nM), and inhibited chloroquine-sensitive and -resistant parasite proliferation (IC50 5–50 µM). Wild-type (WT) parasite IC50 values were up to four-fold lower compared to PfENT1-knockout (pfent1Δ) parasites. pfent1Δ parasite killing showed a delayed-death phenotype not observed with WT. We infer that in parasites, the compounds inhibit both PfENT1 and a secondary target with similar efficacy. The secondary target identity is unknown, but its existence may reduce the likelihood of parasites developing resistance to PfENT1 inhibitors. Our data support the hypothesis that blocking purine transport through PfENT1 may be a novel and compelling approach for antimalarial drug development.
Acetylcholinesterase (AChE) that has been covalently inhibited by organophosphate compounds (OPCs), such as nerve agents and pesticides, has traditionally been reactivated by using nucleophilic oximes. There is, however, a clearly recognized need for new classes of compounds with the ability to reactivate inhibited AChE with improved in vivo efficacy. Here we describe our discovery of new functional groups—Mannich phenols and general bases—that are capable of reactivating OPC-inhibited AChE more efficiently than standard oximes and we describe the cooperative mechanism by which these functionalities are delivered to the active site. These discoveries, supported by preliminary in vivo results and crystallographic data, significantly broaden the available approaches for reactivation of AChE.
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