The DNA polymerase from the bacteriophage T4 is part of a multienzyme complex required for the synthesis of DNA. As a first step in understanding the contributions of individual proteins to the dynamic properties of the complex, e.g., turnover, processivity, and fidelity of replication, the minimal kinetic schemes for the polymerase and exonuclease activities of the gene 43 protein have been determined by pre-steady-state kinetic methods and fit by computer simulation. A DNA primer/template (13/20-mer) was used as substrate; duplexes that contained more single-strand DNA resulted in nonproductive binding of the polymerase. The reaction sequence features an ordered addition of 13/20-mer followed by dATP to the T4 enzyme (dissociation constants of 70 nM and 20 microM) followed by rapid conversion (400 s-1) of the T4.13/20-mer.dATP complex to the T4.14/20-mer.PPi product species. A slow step (2 s-1) following PPi release limits a single turnover, although this step is bypassed in multiple incorporations (13/20-mer-->17/20-mer) which occur at rates > 400 s-1. Competition between correct versus incorrect nucleotides relative to the template strand indicates that the dissociation constants for the incorrect nucleotides are at millimolar values, thus providing evidence that the T4 polymerase, like the T7 but unlike the Klenow fragment polymerases, discriminates by factors > 10(3) against misincorporation in the nucleotide binding step. The exonuclease activity of the T4 enzyme requires an activation step, i.e., T4.DNA-->T4.(DNA)*, whose rate constants reflect whether the 3'-terminus of the primer is matched or mismatched; for matched 13/20-mer the constant is 1 s-1, and for mismatched 13T/20-mer, 5 s-1. Evidence is presented from crossover experiments that this step may represent a melting of the terminus of the duplex, which is followed by rapid exonucleolytic cleavage (100s-1). In the presence of the correct dNTP, primer extension is the rate-limiting step rather than a step involving travel of the duplex between separated exonuclease and polymerase sites. Since the rate constant for 13/20-mer or 13T/20-mer dissociation from the enzyme is 6 or 8 s-1 and competes with that for activation, the exonucleolytic editing by the enzyme alone in a single pass is somewhat inefficient (5 s-1/(8 s-1+5 s-1)), ca. 40%. Consequently, a major role for the accessory proteins may be to slow the rate of enzyme.substrate dissociation, thereby increasing overall fidelity and processivity.

This paper describes the development of a novel microfluorimetric assay to measure the inhibition of Plasmodium falciparum based on the detection of parasitic DNA by intercalation with PicoGreen. The method was used to determine parasite inhibition profiles and 50% inhibitory concentration values of known or potential antimalarial drugs. Values for parasite inhibition with known anti-malarial drugs using the PicoGreen assay were comparable with those determined by the standard method based upon the uptake of 3H-hypoxanthine and the Giemsa stain microscopic technique. The PicoGreen assay is rapid, sensitive, reproducible, easily interpreted, and ideally suited for screening of large numbers of samples for anti-malarial drug development.

Bacteriophage T4 DNA polymerase has a proofreading 3' -+ 5' exonuclease that plays an important role in maintaining the accuracy of DNA replication. We have constructed a T4 DNA polymerase deficient in this exonuclease by converting Asp-219 to Ala. The exonuclease activity of the mutant T4 DNA polymerase has been reduced by a factor of at least 107, but it retains a polymerase activity whose kinetic parameters, k at, Kd DNA, and Kd dATP, are very (17-20). We wished to construct a mutation in T4 DNA polymerase that would remove this exonuclease activity, without altering the polymerase activity, in order to evaluate the role of the exonuclease in maintaining the fidelity of DNA replication and to facilitate kinetic studies of the mechanism by which T4 DNA polymerase alone catalyzes DNA synthesis. Previous studies from one of our laboratories (21) to define a minimal kinetic scheme for T4 DNA polymerase (Scheme I) were complicated by the vigorous exonuclease activity of this enzyme. Exonucleasedeficient enzymes of T7 DNA polymerase (22, 23) and E. coli DNA polymerase I (Klenow fragment) (24) have played integral roles in the delineation of their kinetic mechanisms.The active site for exonuclease must be toward the N terminus ofT4 polymerase, since the protein made by the B22 amber mutant, missing the C-terminal 20% of the intact protein, retains an altered exonuclease but lacks the polymerase activity of the wild-type enzyme (25). Analysis of the crystal structure of the Klenow fragment of E. coli polymerase I and the enzymatic activities of its mutants indicate that Asp-424 is involved in coordination of a metal ion and is essential for exonucleolytic cleavage (26-29). Amino acid sequence comparisons (6) have suggested that Asp-219 in T4 DNA polymerase is in an analogous position. In this report, we describe the construction and initial characterization of a T4 DNA polymerase mutant with alanine replacing Asp-219. We show that this polymerase mutant has no measurable exonuclease, but retains a polymerase activity whose kinetic parameters are very close to those of the wild type. Bacteriophage T4 with the mutant polymerase gene have a markedly increased mutation frequency.MATERIALS AND METHODS

As part of the Panama International Cooperative Biodiversity Groups (ICBG) project, two new (2, 4) and two known (1, 3) linear alkynoic lipopeptides have been isolated from a Panamanian strain of the marine cyanobacterium Lyngbya majuscula. Carmabin A (1), dragomabin (2), and dragonamide A (3) showed good antimalarial activity (IC 50 4.3, 6.0, and 7.7 μM, respectively) whereas the non-aromatic analog, dragonamide B (4), was inactive. The planar structures of all four compounds were determined by NMR spectroscopy in combination with mass spectrometry, and their stereoconfigurations were established by chiral HPLC and by comparison of their optical rotations and NMR data with literature values.Artemisinin Combination Treatments (ACTs) for falciparum malaria are currently the only first-line antimalarial drugs amenable to widespread use against all chloroquine-resistant malaria parasites. 1 However, their effective distribution to combat malaria in economically disadvantaged regions could require an annual global subsidy of $300-500 million. 2 Furthermore, in the event of successful widespread use of the artemisinins, the development of resistance to these drugs before effective replacements or alternatives are at hand is a cause for profound concern. Therefore, the development of new classes of antimalarial drugs remains an enormous challenge and is a focus of many collaborative research efforts, including the International Cooperative Biodiversity Groups project in Panama, which investigates Panamanian terrestrial plants, endophytes and marine organisms as sources of tropical disease treatments. 3 As part of this program, we have been investigating marine cyanobacteria as a source of antimalarial agents, and found that the organic extracts of a red Panamanian strain of the marine cyanobacterium Lyngbya majuscula were active against chloroquine-resistant Plasmodium falciparum. To the best of our knowledge, there are only three reports of marine cyanobacterial metabolites isolated with antimalarial activity. Most recently, members of our * To whom correspondence should be addressed. Tel: 541 737 5808. Fax: 541 737 3999. E-mail: kerry.mcphail@oregonstate.edu. Supporting Information Available: 1 H and 13 C NMR spectra in CDCl 3 for carmabin A (1), dragomabin (2), dragonamide A (3) and dragonamide B (4). Results and DiscussionThe organic extracts of two collections of L. majuscula from different sites in Bocas del Toro, Panama (2002 and2003) showed significant activity against chloroquine-resistant Plasmodium falciparum (IC 50 = 6 and 26 μg/mL). Crude fractionation by normal-phase vacuum-liquid chromatography (NP-VLC) of the most active collection from Isla Bastimentos (Bocas del Toro, 2002) produced two relatively polar fractions (100% EtOAc and 25% MeOH-EtOAc) with good antimalarial activity (<2 and 1 μg/mL, respectively). Solid-phase extraction (SPE) and reversed-phase HPLC of the 25% MeOH-EtOAc NP-VLC fraction yielded carmabin A (1) and dragomabin (2) as the active components.Structure elucidation of li...