The generation of a double-strand break in the Saccharomyces cerevisiae genome is a potentially catastrophic event that can induce cell-cycle arrest or ultimately result in loss of cell viability. The repair of such lesions is strongly dependent on proteins encoded by the RAD52 epistasis group of genes (RAD50-55, RAD57, MRE11, XRS2), as well as the RFA1 and RAD59 genes. rad52 mutants exhibit the most severe phenotypic defects in double-strand break repair, but almost nothing is known about the biochemical role of Rad52 protein. Rad51 protein promotes DNA strand exchange and acts similarly to RecA protein. Yeast Rad52 protein interacts with Rad51 protein, binds single-stranded DNA and stimulates annealing of complementary single-stranded DNA. We find that Rad52 protein stimulates DNA strand exchange by targeting Rad51 protein to a complex of replication protein A (RPA) with single-stranded DNA. Rad52 protein affects an early step in the reaction, presynaptic filament formation, by overcoming the inhibitory effects of the competitor, RPA. Furthermore, stimulation is dependent on the concerted action of both Rad51 protein and RPA, implying that specific protein-protein interactions between Rad52 protein, Rad51 protein and RPA are required.

Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN), the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycero)phosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycero)phosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid-enriched late endosomes, DEN travels through acidified early endosomes, but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid-dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. Currently there are neither vaccines nor effective therapies for DEN, and the essential role of the newly identified DEN-bis(monoacylglycero)phosphate interactions in viral genome escape from the endosome suggests a novel target for drug design.

Protein-promoted DNA strand exchange requires formation of an active presynaptic complex between the DNA-pairing protein and single-stranded DNA (ssDNA). Formation of such a contiguous filament is stimulated by a ssDNA-binding protein. Here, the effects of replication protein A (RPA) on presynaptic complex formation and DNA strand exchange activities of Rad51 protein were examined. Presynaptic complex formation was assessed by measuring ATP hydrolysis. With X174 ssDNA, the ATPase activity of Rad51 protein is stimulated ϳ1.4-fold by RPA, provided that Rad51 protein is in excess of the ssDNA concentration; otherwise, RPA inhibits ATPase activity. In contrast, with ssDNA devoid of secondary structure (poly(dT), poly(dA), poly(dI), and etheno-M13 DNA), RPA does not stimulate the already elevated ATPase activity of Rad51 protein, but inhibits activity at low Rad51 protein concentrations. These results suggest that Rad51 protein and RPA exclude one another from ssDNA by competing for the same binding sites and that RPA exerts its effect on presynaptic complex formation by eliminating secondary structure to which Rad51 protein is bound nonproductively. DNA strand exchange catalyzed by Rad51 protein is also greatly stimulated by RPA. The optimal stoichiometry for stimulation is ϳ20 -30 nucleotides of ssDNA/RPA heterotrimer. The ssDNA-binding protein of Escherichia coli can substitute for RPA, showing that the role of RPA is not specific. We conclude that RPA affects both presynaptic complex formation and DNA strand exchange via changes in DNA structure, employing the same mechanism used by the ssDNA-binding protein to effect change in E. coli RecA protein activity.The RAD51 gene of Saccharomyces cerevisiae, a member of the RAD52 epistasis group, is required for mitotic and meiotic recombination (for a review, see Ref. 1). Cells deficient in RAD51 function are sensitive to x-ray irradiation or DNAalkylating agents, suggesting that this gene is required for repair of double-strand DNA breaks (2). Since formation of meiosis-specific double-strand DNA breaks is not inhibited in rad51 cells, RAD51 seems to function after formation of the break in meiotic recombination. The RAD51 sequence is conserved in a wide variety of eucaryotic organisms, suggesting that it is important to cellular function in eucaryotes (3). Rad51 protein has homology to Escherichia coli RecA protein (2, 4, 5). Furthermore, image reconstruction from electron micrographs of complexes of Rad51 protein and double-stranded DNA (dsDNA) 1 confirmed this similarity and showed that the threedimensional structure of the Rad51 protein-DNA filament is similar to the equivalent RecA protein-dsDNA complex (6). Finally, Rad51 protein from S. cerevisiae has single-stranded DNA (ssDNA)-dependent ATPase activity and promotes ATPdependent DNA strand exchange (7,8).RecA protein plays a central role in genetic recombination in E. coli (for reviews, see Refs. 9 -13). In vitro analyses have revealed that in the presence of ATP, RecA protein binds to ssDNA to form a nucleopro...

Cationic cell-penetrating peptides (CPPs) are a promising vehicle for the delivery of macromolecular drugs. Although many studies have indicated that CPPs enter cells by endocytosis, the mechanisms by which they cross endosomal membranes remain elusive. On the basis of experiments with liposomes, we propose that CPP escape into the cytosol is based on leaky fusion (i.e., fusion associated with the permeabilization of membranes) of the bis(monoacylglycero)phosphate (BMP)-enriched membranes of late endosomes. In our experiments, prototypic CPP HIV-1 TAT peptide did not interact with liposomes mimicking the outer leaflet of the plasma membrane, but it did induce lipid mixing and membrane leakage as it translocated into liposomes mimicking the lipid composition of late endosome. Both membrane leakage and lipid mixing depended on the BMP content and were promoted at acidic pH, which is characteristic of late endosomes. Substitution of BMP with its structural isomer, phosphatidylglycerol (PG), significantly reduced both leakage of the aqueous probe from liposomes and lipid mixing between liposomes. Although affinity of binding to TAT was similar for BMP and PG, BMP exhibited a higher tendency to support the inverted hexagonal phase than PG. Finally, membrane leakage and peptide translocation were both inhibited by inhibitors of lipid mixing, further substantiating the hypothesis that cationic peptides cross BMP-enriched membranes by inducing leaky fusion between them.

Summary HIV-1 entry into host cells starts with interactions between the viral envelope glycoprotein (Env) and cellular CD4 receptors and coreceptors. Previous work has suggested that efficient HIV entry also depends on intracellular signaling but this remains controversial. Here we report that formation of the pre-fusion Env–CD4–coreceptor complexes triggers non-apoptotic cell surface exposure of the membrane lipid phosphatidylserine (PS). HIV-1-induced PS redistribution depends on Ca2+ signaling triggered by Env-coreceptor interactions and involves the lipid scramblase TMEM16F. Externalized PS strongly promotes Env-mediated membrane fusion and HIV-1 infection. Blocking externalized PS or suppressing TMEM16F inhibited Env-mediated fusion. Exogenously added PS promoted fusion, with fusion dependence on PS being especially strong for cells with low surface density of coreceptors. These findings suggest that cell-surface PS acts as an important cofactor that promotes the fusogenic restructuring of pre-fusion complexes and likely focuses the infection on cells conducive to PS signaling.

from Saccharomyces cerevisiae apparently allows DNA heteroduplex extension to occur in either direction (Sung The repair of potentially lethal DNA double-stranded and Robberson, 1995;Namsaraev and Berg, 1998). breaks (DSBs) by homologous recombination requiresAnother eukaryotic homolog, human Rad51 protein, shows processing of the broken DNA into a resected DNA a preferential 3Ј→5Ј polarity for heteroduplex extension, duplex with a protruding 3Ј-single-stranded DNA which is opposite to the direction displayed by the RecA (ssDNA) tail. Accordingly, the canonical models for protein (Baumann and West, 1999). Given the structural DSB repair require invasion of an intact homologous similarities of the nucleoprotein filaments formed by DNA template by the 3Ј-end of the ssDNA, a characterthe eukaryotic Rad51 proteins and RecA protein, this istic that the bacterial pairing protein RecA possesses.difference in the directionality of heteroduplex extension Unexpectedly, we find that for the eukaryotic homolog, is intriguing. Like RecA protein (Stasiak and Di Capua, Rad51 protein, the 5Ј-end of ssDNA is more invasive 1982), both human and yeast Rad51 proteins form helical than the 3Ј-end. This pairing bias is unaffected by filaments with~6.2 protein monomers per turn and a pitch Rad52, Rad54 or Rad55-57 proteins. However, further of~100 Å, stretching the DNA within the filaments by a investigation reveals that, in contrast to RecA protein, factor of 1.5 (Ogawa et al., 1993; Benson et al., 1994). the preferred DNA substrate for Rad51 protein is not Since in the case of RecA protein the polarity of DNA ssDNA but rather dsDNA with ssDNA tails. This heteroduplex extension reflects the underlying polarity of important distinction permits the Rad51 proteins to protein polymerization, as well as the resultant pairing promote DNA strand invasion using either 3Ј-or bias displayed with linear ssDNA (Konforti and Davis, 5Ј-ends with similar efficiency. 1992), the observations with Rad51 protein raise questions Keywords: DNA strand exchange/homologous regarding the homologous pairing preference of the eukarrecombination/joint molecule formation yotic homologs. Therefore, we have examined the pairing bias of Rad51 protein-mediated invasion of supercoiled DNA by linear ssDNA.

There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.

Saccharomyces cerevisiae Rad51 protein is the paradigm for eukaryotic ATP-dependent DNA strand exchange proteins. To explain some of the unique characteristics of DNA strand exchange promoted by Rad51 protein, when compared with its prokaryotic homologue the Escherichia coli RecA protein, we analyzed the DNA binding properties of the Rad51 protein. Rad51 protein binds both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in an ATP-and Mg 2؉ -dependent manner, over a wide range of pH, with an apparent binding stoichiometry of approximately 1 protein monomer per 4 (؎1) nucleotides or base pairs, respectively. Only dATP and adenosine 5-␥-(thiotriphosphate) (ATP␥S) can substitute for ATP, but binding in the presence of ATP␥S requires more than a 5-fold stoichiometric excess of protein. Without nucleotide cofactor, Rad51 protein binds both ssDNA and dsDNA but only at pH values lower than 6.8; in this case, the apparent binding stoichiometry covers the range of 1 protein monomer per 6 -9 nucleotides or base pairs. Therefore, Rad51 protein displays two distinct modes of DNA binding. These binding modes are not inter-convertible; however, their initial selection is governed by ATP binding. On the basis of these DNA binding properties, we conclude that the main reason for the low efficiency of the DNA strand exchange promoted by Rad51 protein in vitro is its enhanced dsDNA-binding ability, which inhibits both the presynaptic and synaptic phases of the DNA strand exchange reaction as follows: during presynapsis, Rad51 protein interacts with and stabilizes secondary structures in ssDNA thereby inhibiting formation of a contiguous nucleoprotein filament; during synapsis, Rad51 protein inactivates the homologous dsDNA partner by directly binding to it.Homologous recombination is a ubiquitous biological process. Elucidation of this process has come mostly from detailed analysis of Escherichia coli RecA protein, a key enzyme in both homologous genetic recombination and recombinational repair of damaged DNA in the eubacteria. RecA-like proteins have been found in a variety of eukaryotes, from yeast to human. The first identified and the most extensively studied eukaryotic RecA protein-analog is the Rad51 protein of Saccharomyces cerevisiae, which is establishing the paradigm for RecA proteinlike functions in the eukaryotes. The RAD51 gene of S. cerevisiae is required for mitotic and meiotic recombination (1) and for the repair of double-strand DNA breaks caused by ionizing irradiation (2). Biochemical studies of Rad51 protein demonstrated that it shares similar properties to RecA protein, including ssDNA 1 -dependent ATPase activity, homologous pairing, and DNA strand exchange activity (3-5), and the formation of helical filaments on both double-stranded DNA (dsDNA) and single-stranded DNA (4, 6) (for review, see Ref. 7).In vitro, the most extensively investigated DNA pairing reaction is the three-strand exchange reaction, in which the pairing of circular ssDNA and homologous dsDNA yields nicked circular dsDNA and li...