We report the x-ray crystal structure of human topoisomerase I covalently joined to double-stranded DNA and bound to the clinically approved anticancer agent Topotecan. Topotecan mimics a DNA base pair and binds at the site of DNA cleavage by intercalating between the upstream (؊1) and downstream (؉1) base pairs. Intercalation displaces the downstream DNA, thus preventing religation of the cleaved strand. By specifically binding to the enzyme-substrate complex, Topotecan acts as an uncompetitive inhibitor. The structure can explain several of the known structure-activity relationships of the camptothecin family of anticancer drugs and suggests that there are at least two classes of mutations that can produce a drug-resistant enzyme. The first class includes changes to residues that contribute to direct interactions with the drug, whereas a second class would alter interactions with the DNA and thereby destabilize the drug-binding site. Eukaryotic DNA topoisomerase I (topo I) is an enzyme that acts to relax supercoils generated during transcription and DNA replication (1). Because of the size of the eukaryotic chromosome, removal of these supercoils can only be accomplished locally by introducing breaks into the DNA helix. Topo I mediates DNA relaxation by creating a transient single-strand break in the DNA duplex. This transient nick allows the broken strand to rotate around its intact complement, effectively removing local supercoils. Strand nicking results from the transesterification of an active-site tyrosine (Tyr-723) at a DNA phosphodiester bond forming a 3Ј-phosphotyrosine covalent enzyme-DNA complex. After DNA relaxation, the covalent intermediate is reversed when the released 5Ј-OH of the broken strand reattacks the phosphotyrosine intermediate in a second transesterification reaction. The rate of religation is normally much faster than the rate of cleavage, and this ensures that the steady-state concentration of the covalent 3Ј-phosphotyrosyl topo I-DNA complex remains low (2).However, a variety of DNA lesions and drugs have been shown to stabilize the covalent 3Ј-phosphotyrosyl intermediate (3). For example, camptothecin (CPT) is a natural product that was originally discovered because of its antitumor activity (4) and was later demonstrated to cause the accumulation of topo I-DNA adducts in vitro and in vivo (5, 6). CPTs bind the covalent 3Ј-phosphotyrosyl intermediate and specifically block DNA religation (7), thus converting topo I into a DNA-damaging agent (8). Topo I is the sole intramolecular target of CPT, and the cytotoxic effects of CPT poisoning are S-phase specific (9). During DNA replication, the replication fork is thought to collide with the ''trapped'' topo I-DNA complexes, resulting in double-strand breaks and ultimately cell death (10).It has been difficult to study the mechanism of CPT activity because the drug acts as an uncompetitive inhibitor and binds only the transient covalent enzyme-substrate complex (7,11). To isolate the covalent topo I-DNA complex, we have used suicide DNA ...

Basic and acidic fibroblast growth factors (FGF's) are potent mitogens for capillary endothelial cells in vitro, stimulate angiogenesis in vivo, and may participate in tissue repair. An oligonucleotide probe for bovine basic FGF was designed from the nucleotide sequence of the amino-terminal exon of bovine acidic FGF, taking into account the 55 percent amino acid sequence homology between the two factors. With this oligonucleotide probe, a full length complementary DNA for basic FGF was isolated from bovine pituitary. Basic FGF in bovine hypothalamus was shown to be encoded by a single 5.0-kilobase messenger RNA; in a human hepatoma cell line, both 4.6- and 2.2-kilobase basic FGF messenger RNA's were present. Both growth factors seem to be synthesized with short amino-terminal extensions that are not found on the isolated forms for which the amino acid sequences have been determined. Neither basic nor acidic FGF has a classic signal peptide.

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SummaryAntigenic diversity has posed a critical barrier to vaccine development against the pathogenic blood-stage infection of the human malaria parasite Plasmodium falciparum. To date, only strain-specific protection has been reported by trials of such vaccines in nonhuman primates. We recently showed that P. falciparum reticulocyte binding protein homolog 5 (PfRH5), a merozoite adhesin required for erythrocyte invasion, is highly susceptible to vaccine-inducible strain-transcending parasite-neutralizing antibody. In vivo efficacy of PfRH5-based vaccines has not previously been evaluated. Here, we demonstrate that PfRH5-based vaccines can protect Aotus monkeys against a virulent vaccine-heterologous P. falciparum challenge and show that such protection can be achieved by a human-compatible vaccine formulation. Protection was associated with anti-PfRH5 antibody concentration and in vitro parasite-neutralizing activity, supporting the use of this in vitro assay to predict the in vivo efficacy of future vaccine candidates. These data suggest that PfRH5-based vaccines have potential to achieve strain-transcending efficacy in humans.