Homologous recombination serves multiple roles in DNA repair that are essential for maintaining genomic stability. We here describe RI-1, a small molecule that inhibits the central recombination protein RAD51. RI-1 specifically reduces gene conversion in human cells while stimulating single strand annealing. RI-1 binds covalently to the surface of RAD51 protein at cysteine 319 that likely destabilizes an interface used by RAD51 monomers to oligomerize into filaments on DNA. Correspondingly, the molecule inhibits the formation of subnuclear RAD51 foci in cells following DNA damage, while leaving replication protein A focus formation unaffected. Finally, it potentiates the lethal effects of a DNA cross-linking drug in human cells. Given that this inhibitory activity is seen in multiple human tumor cell lines, RI-1 holds promise as an oncologic drug. Furthermore, RI-1 represents a unique tool to dissect the network of reaction pathways that contribute to DNA repair in cells.

RAD51 and other members of the RecA family of strand exchange proteins assemble on ssDNA to form presynaptic filaments, which carry out the central steps of homologous recombination. A microplate-based assay was developed for high-throughput measurement of hRAD51 filament formation on ssDNA. With this method, a 10,000 compound library was screened, leading to the identification of a small molecule (RS-1) that enhances hRAD51 binding in a wide range of biochemical conditions. Salt titration experiments showed that RS-1 can enhance filament stability. Ultrastructural analysis of filaments formed on ssDNA showed that RS-1 can increase both protein-DNA complex lengths and the pitch of helical filament turns. RS-1 stimulated hRAD51-mediated homologous strand assimilation (D-loop) activity by at least 5-to 11-fold, depending on the condition. This D-loop stimulation occurred even in the presence of Ca 2؉ or adenylyl-imidodiphosphate, indicating that the mechanism of stimulation was distinct from that conferred by Ca 2؉ and/or inhibition of ATPase. No D-loop activity was observed in the absence of a nucleotide triphosphate cofactor, indicating that the compound does not substitute for this requirement. These results indicate that RS-1 enhances the homologous recombination activity of hRAD51 by promoting the formation of active presynaptic filaments. Cell survival assays in normal neonatal human dermal fibroblasts demonstrated that RS-1 promotes a dose-dependent resistance to the cross-linking chemotherapeutic drug cisplatin. Given that RAD51-dependent recombination is a major determinant of cisplatin resistance, RS-1 seems to function in vivo to stimulate homologous recombination repair proficiency. RS-1 has many potential applications in both research and medical settings.cross-linking chemotherapy ͉ DNA repair ͉ high-throughput screen ͉ recombinase ͉ strand exchange H omologous recombination (HR) has multiple roles in DNA repair, including the repair of double strand breaks (DSBs) and recovery from the replication-blocking lesions formed by DNA cross-linking agents. HR repairs DSBs by locating a homologous stretch of DNA and replicating the missing genetic information from this homologous template. In contrast to DSB repair by nonhomologous end joining, HR repair generally occurs without mutations. Because of this, HR repair is critically important in the maintenance of genomic stability (reviewed in ref. 1). The proposed mechanism for this pathway begins with 5Ј to 3Ј nuclease activity at the DSB, resulting in a 3Ј singlestranded tail. The tail is coated by replication protein A, which is subsequently replaced by a helical filament of RAD51 protein. This displacement of replication protein A by RAD51 seems to be controlled by a number of mediator proteins, which include BRCA2, RAD52, and RAD51 paralogue complexes (2-5). The RAD51-coated 3Ј tail then locates and invades a homologous template of dsDNA. After invasion, templated DNA synthesis initiated at 3Ј ends leads to formation of branched DNA intermediates, which ...

Coronavirus spike (S) proteins are palmitoylated at several cysteine residues clustered near their transmembrane-spanning domains. This is achieved by cellular palmitoyl acyltransferases (PATs), which can modify newly synthesized S proteins before they are assembled into virion envelopes at the intermediate compartment of the exocytic pathway. To address the importance of these fatty acylations to coronavirus infection, we exposed infected cells to 2-bromopalmitate (2-BP), a specific PAT inhibitor. 2-BP profoundly reduced the specific infectivities of murine coronaviruses at very low, nontoxic doses that were inert to alphavirus and rhabdovirus infections. 2-BP effected only two-to fivefold reductions in S palmitoylation, yet this correlated with reduced S complexing with virion membrane (M) proteins and consequent exclusion of S from virions. At defined 2-BP doses, underpalmitoylated S proteins instead trafficked to infected cell surfaces and elicited cell-cell membrane fusions, suggesting that the acyl chain adducts are more critical to virion assembly than to S-induced syncytial developments. These studies involving pharmacologic inhibition of S protein palmitoylation were complemented with molecular genetic analyses in which cysteine acylation substrates were mutated. Notably, some mutations (C1347F and C1348S) did not interfere with S incorporation into virions, indicating that only a subset of the cysteine-rich region provides the essential S-assembly functions. However, the C1347F/C1348S mutant viruses exhibited relatively low specific infectivities, similar to virions secreted from 2-BP-treated cultures. Our collective results indicate that the palmitate adducts on coronavirus S proteins are necessary in assembly and also in positioning the assembled envelope proteins for maximal infectivity.Palmitoylation is a common posttranslational modification that can influence protein trafficking and protein-protein and protein-membrane interactions. The hydrophobic acyl chains are linked in thioesterification reactions to cysteine residues residing in the cytoplasmic tails of several viral membrane glycoproteins, including the influenza virus hemagglutinin, paramyxovirus F, vesicular stomatitis virus (VSV) G, Sindbis virus (SV) E1, retrovirus Env, baculovirus gp64, and coronavirus spike (S). The importance of these lipid modifications to viral glycoprotein structure is not precisely known; however, it is reasonable to assume that they act to position cytoplasmic tails at juxtamembrane locations, thereby contributing a membrane anchoring that is secondary to protein transmembrane spans. This tethering to the cytoplasmic leaflets of lipid bilayers may have several distinct functional ramifications. There is evidence that palmitate adducts alter protein transport in the cellular exocytic pathway (34), assist in clustering glycoproteins into lipid microdomains (5, 55), and enforce membrane anchoring during the refolding events accompanying viral glycoprotein-mediated membrane fusions (50). Given these varied modes by...

The coronavirus assembly process encloses a ribonucleoprotein genome into vesicles containing the lipidembedded proteins S (spike), E (envelope), and M (membrane). This process depends on interactions with membranes that may involve palmitoylation, a common posttranslational lipidation of cysteine residues. To determine whether specific palmitoylations influence coronavirus assembly, we introduced plasmid DNAs encoding mouse hepatitis coronavirus (MHV) S, E, M, and N (nucleocapsid) into 293T cells and found that virus-like particles (VLPs) were robustly assembled and secreted into culture medium. Palmitate adducts predicted on cysteines 40, 44, and 47 of the 83-residue E protein were then evaluated by constructing mutant cDNAs with alanine or glycine codon substitutions at one or more of these positions. Triple-substituted proteins (E.Ts) lacked palmitate adducts. Both native E and E.T proteins localized at identical perinuclear locations, and both copurified with M proteins, but E.T was entirely incompetent for VLP production. In the presence of the E.T proteins, the M protein subunits accumulated into detergent-insoluble complexes that failed to secrete from cells, while native E proteins mobilized M into detergent-soluble secreted forms. Many of these observations were corroborated in the context of natural MHV infections, with native E, but not E.T, complementing debilitated recombinant MHVs lacking E. Our findings suggest that palmitoylations are essential for E to act as a vesicle morphogenetic protein and further argue that palmitoylated E proteins operate by allowing the primary coronavirus assembly subunits to assume configurations that can mobilize into secreted lipid vesicles and virions.Coronavirus-infected cells provide models to investigate protein targeting, subcellular protein transport, protein-lipid and protein-protein interactions, and of multiprotein complex assembly. This is because all of these events take place efficiently and in organized temporal fashion within cells to create infectious, lipid-enveloped virus particles. The essential components of infectious coronavirions include three membrane proteins, S (spike), E (envelope), and M (membrane), along with cytoplasmic N (nucleocapsid). These four proteins, along with newly synthesized monopartite plus-strand RNA virus genomes, congregate at endoplasmic reticulum and Golgi membranes, ultimately remodeling the membranes such that they intrude inward toward organelle lumen and then undergo membrane fission to create enveloped virus vesicles. Accomplishing these morphological changes involves several wellknown interactions. N proteins bind viral RNAs to generate helical ribonucleoprotein (RNP) complexes (27), RNPs in turn bind to the cytoplasmic carboxyl-terminal extensions of M proteins (25, 45), M proteins bind to each other (9), and M and S proteins interact at or near their transmembrane-spanning regions (10,16,57). Subcellular targeting signals in the S, E, and M proteins interacting with presumed cellular factors restrict association...

Homologous recombination (HR) and non-homologous end joining (NHEJ) are alternative pathways of double-strand DNA break repair. We developed a method to quantify the efficiency of DNA repair pathways in the context of cancer therapy. The Recombination Proficiency Score (RPS) utilizes the expression levels for four genes involved in DNA repair pathway preference (RIF1, PARI, RAD51, and Ku80), such that high expression of these genes yields a low RPS. Carcinoma cells with low RPS exhibit HR suppression and frequent DNA copy number alterations, which are characteristic of error-prone repair processes that arise in HR-deficient backgrounds. The RPS system was clinically validated in patients with breast or non-small cell lung carcinomas (NSCLC). Tumors with low RPS were associated with greater mutagenesis, adverse clinical features, and inferior patient survival rates, suggesting that HR suppression plays a central role in promoting the genomic instability that fuels malignant progression. This adverse prognosis associated with low RPS was diminished if NSCLC patients received adjuvant chemotherapy, suggesting that HR suppression and associated sensitivity to platinum-based drugs counteracts the adverse prognosis associated with low RPS. Therefore, RPS may predict which therapies will be effective for individual patients, thereby enabling more personalized oncology care.

RAD51 is the central protein that catalyzes DNA repair via homologous recombination (HR), a process that ensures genomic stability. RAD51 protein is commonly expressed at high levels in cancer cells relative to their non-cancerous precursors. High levels of RAD51 expression can lead to the formation of genotoxic RAD51 protein complexes on undamaged chromatin. We developed a therapeutic approach that exploits this potentially toxic feature of malignancy, using compounds that stimulate the DNA binding activity of RAD51 to promote cancer cell death. A panel of immortalized cell lines was challenged with the RAD51-stimulatory compound RS-1. Resistance to RS-1 tended to occur in cells with higher levels of RAD54L and RAD54B, which are Swi2/Snf2-related translocases known to dissociate RAD51 filaments from double-stranded DNA. In PC3 prostate cancer cells, RS-1 induced lethality was accompanied by the formation of microscopically visible RAD51 nuclear protein foci occurring in the absence of any DNA-damaging treatment. Treatment with RS-1 promoted significant anti-tumor responses in a mouse model, providing proof of principle for this novel therapeutic strategy.

<p>PDF file - 403KB, Supplemental Figure 1. Quantifications of western blots. Supplemental Figure 2. Knockdown of RAD51 in PC3 does not protect cells from the toxicity of ionizing radiation. Supplemental Table 1. Quantifications of statistical significance are displayed for the RS-1 toxicity data that are displayed in Figure 2. Supplemental Methods. Chemical synthesis of compounds.</p>

<p>PDF file - 403KB, Supplemental Figure 1. Quantifications of western blots. Supplemental Figure 2. Knockdown of RAD51 in PC3 does not protect cells from the toxicity of ionizing radiation. Supplemental Table 1. Quantifications of statistical significance are displayed for the RS-1 toxicity data that are displayed in Figure 2. Supplemental Methods. Chemical synthesis of compounds.</p>