V(D)J recombination is instigated by the recombination-activating proteins RAG1 and RAG2, which catalyze site-specific DNA cleavage at the border of the recombination signal sequence (RSS). Although both proteins are required for activity, core RAG1 (the catalytically active region containing residues 384 -1008 of 1040) alone displays binding specificity for the conserved heptamer and nonamer sequences of the RSS. The nonamer-binding region lies near the N terminus of core RAG1, whereas the heptamer-binding region has not been identified. Here, potential domains within core RAG1 were identified using limited proteolysis studies. An iterative procedure of DNA cloning, protein expression, and characterization revealed the presence of two topologically independent domains within core RAG1, referred to as the central domain (residues 528 -760) and the C-terminal domain (residues 761-980). The domains do not include the nonamer-binding region but rather largely span the remaining relatively uncharacterized region of core RAG1. Characterization of macromolecular interactions revealed that the central domain bound to the RSS with specificity for the heptamer and contained the predominant binding site for RAG2. The Cterminal domain bound DNA cooperatively but did not show specificity for either conserved RSS element. This domain was also found to self-associate, implicating it as a dimerization domain within RAG1.The immune system displays remarkable specificity and diversity in its ability to recognize and eliminate foreign antigens. The basis for this immense diversity in many species is a complex rearrangement of the V (variable), D (diversity), and J (joining) gene segments that together encode the variable regions of T cell receptors and immunoglobulins (see Ref. 1 for review). This process, known as V(D)J recombination, requires the activity of a wide array of enzymes and is initiated by the lymphoid-specific recombination-activating proteins RAG1 and RAG2. The RAG proteins guide recombination events to conserved recombination signal sequences (RSSs) 1 that flank the genomic regions to be rearranged. Each RSS consists of a conserved heptamer and nonamer sequence separated by a 12-or 23-base pair spacer, the sequence of which is poorly conserved. Efficient recombination occurs generally between an RSS containing a 12-base pair spacer (12RSS) and one containing a 23-base pair spacer (23RSS), a requirement referred to as the 12/23 rule.The recombination process is often divided into two phases, the first phase of which consists of two distinct enzymatic steps catalyzed by the RAG proteins. The first step involves the binding of a RAG1-RAG2 complex to an RSS and the subsequent generation of a nick between the heptamer and its adjacent coding strand. The resulting 3Ј-OH group then performs a nucleophilic attack on the phosphodiester bond of the opposite strand. The primary products of this transesterification reaction are a covalently sealed hairpin, referred to as the coding end, and a blunt-ended 5Ј phosphorylated ...

RAG1 and RAG2 initiate V(D)J recombination by introducing DNA double strand breaks between each selected gene segment and its bordering recombination signal sequence (RSS) in a two-step mechanism in which the DNA is first nicked, followed by hairpin formation. The RSS consists of a conserved nonamer and heptamer sequence, in which the latter borders the site of DNA cleavage. A region within RAG1, referred to as the central domain (residues 528 -760 of 1040 in the full-length protein), has been shown previously to bind specifically to the double-stranded (ds) RSS heptamer, but with both weak specificity and affinity. However, additional investigations into the RAG1-RSS heptamer interaction are required because the DNA substrate forms intermediate conformations during the V(D)J recombination reaction. These include the nicked and hairpin products, as well as likely base unpairing to produce single-stranded (ss) DNA near the cleavage site. Here, it was determined that although the central domain showed substantially higher binding affinity for ss and nicked versus ds substrate, the interaction with ss RSS was particularly robust. In addition, the central domain bound with greater sequence specificity to the ss RSS heptamer than to the ds form. This study provides important insight into the V(D)J recombination reaction, specifically that significant interaction of the RSS heptamer with RAG1 occurs only after the induction of conformational changes at the RSS heptamer.V(D)J recombination leads to the formation of functional immunoglobulin and T cell receptor genes in developing B and T cells, respectively. Through DNA rearrangement of the immunoglobulin and T cell receptor genetic loci, the genes are assembled by combining selected gene segments termed variable (V), joining (J), and at some loci, diversity (D). The variability of the assembly process in each developing lymphocyte yields an immune system that contains a repertoire of antigen receptors with an array of binding specificities (1, 2).The first phase of V(D)J recombination consists of site-specific DNA cleavage steps adjacent to selected gene segments and requires the lymphoid-specific recombination activating proteins, RAG1 and RAG2 (1, 2). Together the RAG proteins are directed to potential DNA cleavage sites by recognition of the recombination signal sequence (RSS), 1 which flanks each gene segment. The RSS contains a conserved heptamer and nonamer sequence separated by either 12 (12-RSS) or 23 (23-RSS) base pairs of poorly conserved DNA. Successful assembly of two gene segments requires that the segments are adjoined to dissimilar RSSs, a requirement referred to as the 12/23 rule. The RAG proteins cleave at each selected RSS to first nick the double-stranded DNA between the RSS heptamer and the bordering antigen receptor gene segment. The result is a 3Ј-OH group that executes a nucleophilic attack on the opposite strand, generating a covalently closed hairpin at the coding end and a signal end that is blunt-ended at the 5Ј-end of the RSS heptamer. The ...

Background Trichomonas vaginalis is the most prevalent non-viral sexually transmitted infection. We evaluated the efficacy and safety of secnidazole vs. placebo in women with trichomoniasis. Methods Women with trichomoniasis, confirmed by a positive T. vaginalis culture, were randomized to single-dose oral secnidazole 2g or placebo. The primary endpoint was microbiological test of cure (TOC) by culture 6–12 days after dosing. At the TOC visit, participants were given the opposite treatment. They were followed for resolution of infection afterward and offered treatment at subsequent visits, if needed. Fifty patients per group (N=100) provided ~95% power to detect a statistically significant difference between treatment groups. Results Between April 2019 and March 2020, 147 women enrolled at 10 US sites. The modified intent-to-treat (mITT) population included 131 randomized patients (64/67, in secnidazole/placebo). Cure rates were significantly higher in the secnidazole vs. placebo group (92.2% [95% CI: 82.7–97.4] vs. 1.5% [95% CI: 0.0–8.0]) for the mITT population and for the per-protocol population (94.9% [95% CI: 85.9–98.9]) vs. 1.7% [95% CI: 0.0–8.9]). Cure rates were 100% (4/4) in women with HIV and 95.2% (20/21) in women with bacterial vaginosis (BV). Secnidazole was generally well tolerated. The most frequently reported treatment-emergent adverse events (TEAEs) were vulvovaginal candidiasis and nausea (each 2.7%). No serious TEAEs were observed. Conclusion A single oral 2g dose of secnidazole was associated with significantly higher microbiological cure rates vs. placebo, supporting a role for secnidazole in treating women with trichomoniasis, including those with HIV and/or BV.

BackgroundThe repertoire of the antigen-binding receptors originates from the rearrangement of immunoglobulin and T-cell receptor genetic loci in a process known as V(D)J recombination. The initial site-specific DNA cleavage steps of this process are catalyzed by the lymphoid specific proteins RAG1 and RAG2. The majority of studies on RAG1 and RAG2 have focused on the minimal, core regions required for catalytic activity. Though not absolutely required, non-core regions of RAG1 and RAG2 have been shown to influence the efficiency and fidelity of the recombination reaction.ResultsUsing a partial proteolysis approach in combination with bioinformatics analyses, we identified the domain boundaries of a structural domain that is present in the 380-residue N-terminal non-core region of RAG1. We term this domain the Central Non-core Domain (CND; residues 87-217).ConclusionsWe show how the CND alone, and in combination with other regions of non-core RAG1, functions in nuclear localization, zinc coordination, and interactions with nucleic acid. Together, these results demonstrate the multiple roles that the non-core region can play in the function of the full length protein.