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Background: The invariant TCR ζ/CD247 homodimer is crucial for TCR/CD3 expression and signaling through its 3 immunoreceptor tyrosine-based activation motifs (ITAMs). Homozygous null mutations in CD247 lead to immunodeficiency, while carriers exhibit 50% reduced surface CD3. It is unclear whether carriers of other CD247 variants show dominant-negative effects., Objective: We sought to analyze and model the potential impact on T-cell receptor (TCR) expression and function of heterozygous nonsense CD247 mutations found in patients with signs of immunodeficiency or autoimmunity., Methods: Jurkat T cells, either wild-type (WT) or CRISPR/Cas9-edited CD247-deficient (ZKO), were lentivirally transduced with WT CD247 or mutations ablating 1 (Q142X), 2 (Q101X), or 3 (Q70X) ITAMs., Results: Three patients from unrelated families were studied. Two heterozygous nonsense CD247 mutations were identified (p.Y152X and p.Q101X), which affected ITAM-3 and ITAM-2 and ITAM-3, respectively. Both mutations were associated with low surface CD3 expression and normal intracellular CD247 levels using a transmembrane-specific antibody, but very low intracellular CD247 levels using an ITAM-3-specific one, suggesting the presence of truncated variants in T cells. Transduction of the mutations lacking 1, 2, or 3 ITAMs into ZKO cells could not restore normal surface CD3 expression (only 60%, 22%, and 10%, respectively), whereas in WT cells, normal surface CD3 expression was reduced (to 39%, 19%, and 9% of normal levels), and both effects were dependent on ITAM number. All 6 transfectants showed reduced CD69 induction (25% to 50%), indicating that they were unable to signal downstream properly, neither isolated nor associated with WT CD247., Conclusions: Our results suggest that CD247 variants lacking ITAMs due to nonsense, but not null, mutations are defective for normal TCR assembly and exert a dominant-negative effect on TCR expression and signaling in vitro. This, in turn, may correlate with clinical features in vivo., Competing Interests: Disclosure statement This work was supported by grants from the Ministerio de Economía y Competitividad (MINECO PID2021-125501OB-I00 and RTI2018-095673-B-I00) and Asociación Española Contra el Cáncer (AECC PROYE20084REGU). A.C.B. was supported by Complutense University scholarship (CT27/16 and CT31/21). R.F.M. and D.C.A. were supported by the Spanish Ministry of Science, Innovation and Universities (FPU19/03136 and PRE2019-088150). P.P.C. was supported by Juan de la Cierva-Incorporación fellowship (IJCI-2014-19262). Disclosure of potential conflict of interest: The authors declare that they have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-α T cell receptor (pre-TCRα) expression. Low circulating naive αβ T cell counts at birth persisted over time, with normal memory αβ and high γδ T cell counts. Their TCRα repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue αβ T cell development. Only a minority of these individuals were sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naive αβ T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αβ T cells, autoimmune conditions were more frequent in these patients compared with the general population.

Bacillus subtilis cells respond to double strand breaks (DSBs) with an ordered recruitment of repair proteins to the site lesion, being RecN one of the first responders. In B. subtilis, one of the responses to DSBs is to increase RecN expression rather than modifying its turnover rate. End-processing activities and the RecA protein itself contribute to increase RecN levels after DNA DSBs. RecO is required for RecA filament formation and full SOS induction, but its absence did not significantly affect RecN expression. Neither the absence of LexA nor the phosphorylation state of RecA or SsbA significantly affect RecN expression levels. These findings identify two major mechanisms (SOS and DSB response) used to respond to DSBs, with LexA required for one of them (SOS response). The DSB response, which requires end-processing and RecA or short RecO-independent RecA filaments, highlights the importance of guarding genome stability by modulating the DNA damage responses., (Copyright © 2013 Elsevier B.V. All rights reserved.)

All organisms rely on integrated networks to repair DNA double-strand breaks (DSBs) in order to preserve the integrity of the genetic information, to re-establish replication, and to ensure proper chromosomal segregation. Genetic, cytological, biochemical and structural approaches have been used to analyze how Bacillus subtilis senses DNA damage and responds to DSBs. RecN, which is among the first responders to DNA DSBs, promotes the ordered recruitment of repair proteins to the site of a lesion. Cells have evolved different mechanisms for efficient end processing to create a 3'-tailed duplex DNA, the substrate for RecA binding, in the repair of one- and two-ended DSBs. Strand continuity is re-established via homologous recombination (HR), utilizing an intact homologous DNA molecule as a template. In the absence of transient diploidy or of HR, however, two-ended DSBs can be directly re-ligated via error-prone non-homologous end-joining. Here we review recent findings that shed light on the early stages of DSB repair in Firmicutes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A silicon nanowire-based biosensor has been designed and applied for label-free and ultrasensitive detection of the early stage of recombinational DNA repair by RecA protein. Silicon nanowires transistors were fabricated by atomic force microscopy nanolithography and integrated into a microfluidic environment. The sensor operates by measuring the changes in the resistance of the nanowire as the biomolecular reactions proceed. We show that the nanoelectronic sensor can detect and differentiate several steps in the binding of RecA to a single-stranded DNA filament taking place on the nanowire-aqueous interface. We report relative changes in the resistance of 3.5% which are related to the interaction of 250 RecA·single-stranded DNA complexes. Spectroscopy data confirm the presence of the protein-DNA complexes on the functionalized silicon surfaces., (© 2012 American Chemical Society)

Bacillus subtilis pnpA gene product, polynucleotide phosphorylase (PNPase), is involved in double-strand break (DSB) repair via homologous recombination (HR) or non-homologous end-joining (NHEJ). RecN is among the first responders to localize at the DNA DSBs, with PNPase facilitating the formation of a discrete RecN focus per nucleoid. PNPase, which co-purifies with RecA and RecN, was able to degrade single-stranded (ss) DNA with a 3' → 5' polarity in the presence of Mn(2+) and low inorganic phosphate (Pi) concentration, or to extend a 3'-OH end in the presence dNDP · Mn(2+). Both PNPase activities were observed in evolutionarily distant bacteria (B. subtilis and Escherichia coli), suggesting conserved functions. The activity of PNPase was directed toward ssDNA degradation or polymerization by manipulating the Pi/dNDPs concentrations or the availability of RecA or RecN. In its dATP-bound form, RecN stimulates PNPase-mediated polymerization. ssDNA phosphorolysis catalyzed by PNPase is stimulated by RecA, but inhibited by SsbA. Our findings suggest that (i) the PNPase degradative and polymerizing activities might play a critical role in the transition from DSB sensing to end resection via HR and (ii) by blunting a 3'-tailed duplex DNA, in the absence of HR, B. subtilis PNPase might also contribute to repair via NHEJ.

In the presence of Mn(2+), an activity in a preparation of purified Bacillus subtilis RecN degrades single-stranded (ss) DNA with a 3' --> 5' polarity. This activity is not associated with RecN itself, because RecN purified from cells lacking polynucleotide phosphorylase (PNPase) does not show the exonuclease activity. We show here that, in the presence of Mn(2+) and low-level inorganic phosphate (P(i)), PNPase degrades ssDNA. The limited end-processing of DNA is regulated by ATP and is inactive in the presence of Mg(2+) or high-level P(i). In contrast, the RNase activity of PNPase requires Mg(2+) and P(i), suggesting that PNPase degradation of RNA and ssDNA occur by mutually exclusive mechanisms. A null pnpA mutation (DeltapnpA) is not epistatic with Delta recA, but is epistatic with DeltarecN and Delta ku, which by themselves are non-epistatic. The addA5, Delta recO, Delta recQ (Delta recJ), Delta recU and Delta recG mutations (representative of different epistatic groups), in the context of DeltapnpA, demonstrate gain- or loss-of-function by inactivation of repair-by-recombination, depending on acute or chronic exposure to the damaging agent and the nature of the DNA lesion. Our data suggest that PNPase is involved in various nucleic acid metabolic pathways, and its limited ssDNA exonuclease activity plays an important role in RecA-dependent and RecA-independent repair pathways.

Genetic and cytological evidences suggest that Bacillus subtilis RecN acts prior to and after end-processing of DNA double-strand ends via homologous recombination, appears to participate in the assembly of a DNA repair centre and interacts with incoming single-stranded (ss) DNA during natural transformation. We have determined the architecture of RecN-ssDNA complexes by atomic force microscopy (AFM). ATP induces changes in the architecture of the RecN-ssDNA complexes and stimulates inter-complex assembly, thereby increasing the local concentration of DNA ends. The large CII and CIII complexes formed are insensitive to SsbA (counterpart of Escherichia coli SSB or eukaryotic RPA protein) addition, but RecA induces dislodging of RecN from the overhangs of duplex DNA molecules. Reciprocally, in the presence of RecN, RecA does not form large RecA-DNA networks. Based on these results, we hypothesize that in the presence of ATP, RecN tethers the 3'-ssDNA ends, and facilitates the access of RecA to the high local concentration of DNA ends. Then, the resulting RecA nucleoprotein filaments, on different ssDNA segments, might promote the simultaneous genome-wide homology search.

Plasmodium vivax merozoite surface protein 4 (PvMSP4) has been considered to be a promising malarial vaccine candidate. The antigenic diversity displayed by parasite populations is one of the main factors limiting the efficacy of asexual-stage anti-malarial vaccine candidates. The present study is the first characterising PvMSP4 polymorphism. P. vivax isolates were collected from endemic areas in Colombia and diversity and selection pattern studies were carried out. Overall conservation in this protein was remarkable. Changes were only found in exons I and II, the former only having single nucleotide polymorphisms (SNPs) whilst the latter showed variations in tandem repeat number caused by exon II slippage. The remaining regions (EGF-like domain, GPI anchor and intron) were completely conserved. Selection and neutrality tests carried out over variant exons indicated negative selective forces acting on them. No evidence of intragenic recombination was found. The strong conservation displayed in this molecule by isolates from geographically different regions (Colombia, Salvador and Thailand) stresses its potential importance as a candidate for a vaccine against P. vivax malaria.

The MHC class I molecule plays an important role in immune response, pathogen recognition and response against vaccines and self- versus non-self-recognition. Studying MHC class I characteristics thus became a priority when dealing with Aotus to ensure its use as an animal model for biomedical research. Isolation, cloning and sequencing of exons 1-8 from 27 MHC class I alleles obtained from 13 individuals classified as belonging to three owl monkey species (A. nancymaae, A. nigriceps and A. vociferans) were carried out to establish similarities between Aotus MHC class I genes and those expressed by other New and Old World primates. Six Aotus MHC class I sequence groups (Ao-g1, Ao-g2, Ao-g3, Ao-g4, Ao-g5 and Ao-g6) weakly related to non-classical Catarrhini MHC were identified. An allelic lineage was also identified in one A. nancymaae and two A. vociferans monkeys, exhibiting a high degree of conservation, negative selection along the molecule and premature termination of the open reading frame at exon 5 (Ao-g5). These sequences' high conservation suggests that they more likely correspond to a soluble form of Aotus MHC class I molecules than to a new group of processed pseudogenes. Another group, named Ao-g6, exhibited a strong relationship with Catarrhini's classical MHC-B-C loci. Sequence evolution and variability analysis indicated that Aotus MHC class I molecules experience inter-locus gene conversion phenomena, contributing towards their high variability.

The population of malaria-causing parasites is characterized by great genetic diversity. Knowledge of the polymorphism generation mechanism is a central issue for developing effective vaccines against malaria and understanding the parasite population structure. Plasmodium vivax genetic diversity has been explained in terms of two major factors: natural selection and intragenic recombination. A modular organization was found within P. vivax Duffy binding protein in the present work. Four Colombian isolates have identical sequences to Salvador-1 strain amongst dpb regions III-VI analysed, suggesting a high identity between Central and South American isolates. Geographically clustered sectors, corresponding to cysteine-rich regions (II and VI), show a high sequence diversity that could reflect a possible immune response evasion mechanism; both positive and negative selection were detected in these regions. In contrast, other dbp gene regions display a non-geographical clustering pattern, lower sequence diversity and predominant negative selective pressure. Recombination was homogeneously detected all along the molecule. These findings suggest that diversification vs. homogenizing forces, drive dbp gene evolution and determine its mosaic region organization.