The immunopathological landscape of human pre-TCRα deficiency: From rare to common variants.

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-α T cell receptor (pre-TCRα) expression. Low circulating naive αß T cell counts at birth persisted over time, with normal memory αß and high γδ T cell counts. Their TCRα repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue αß 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 αß T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αß T cells, autoimmune conditions were more frequent in these patients compared with the general population.

CD3G or CD3D Knockdown in Mature, but Not Immature, T Lymphocytes Similarly Cripples the Human TCRαß Complex.

The human αß T-cell receptor (TCR) is composed of a variable heterodimer (TCRαß) and three invariant dimers (CD3γε, CD3δε, and ζζ/CD2472). The role of each invariant chain in the stepwise interactions among TCR chains along the assembly is still not fully understood. Despite the high sequence homology between CD3γ and CD3δ, the clinical consequences of the corresponding immunodeficiencies (ID) in humans are very different (mild and severe, respectively), and mouse models do not recapitulate findings in human ID. To try to understand such disparities, we stably knocked down (KD) CD3D or CD3G expression in the human Jurkat T-cell line and analyzed comparatively their impact on TCRαß assembly, transport, and surface expression. The results indicated that TCR ensembles were less stable and CD3ε levels were lower when CD3γ, rather than CD3δ, was scarce. However, both defective TCR ensembles were strongly retained in the ER, lacked ζζ/CD2472, and barely reached the T-cell surface (<11% of normal controls) in any of the CD3 KD cells. This is in sharp contrast to human CD3γ ID, whose mature T cells express higher levels of surface TCR (>30% vs. normal controls). CD3 KD of human T-cell progenitors followed by mouse fetal thymus organ cultures showed high plasticity in emerging immature polyclonal T lymphocytes that allowed for the expression of significant TCR levels which may then signal for survival in CD3γ, but not in CD3δ deficiency, and explain the immunological and clinical disparities of such ID cases.

Bacillus subtilis RarA Acts as a Positive RecA Accessory Protein.

Ubiquitous RarA AAA+ ATPases play crucial roles in the cellular response to blocked replication forks in pro- and eukaryotes. Here, we provide evidence that absence of RarA reduced the viability of ΔrecA, ΔrecO, and recF15 cells during unperturbed growth. The rarA gene was epistatic to recO and recF genes in response to H2O2- or MMS-induced DNA damage. Conversely, the inactivation of rarA partially suppressed the HR defect of mutants lacking end-resection (ΔaddAB, ΔrecJ, ΔrecQ, ΔrecS) or branch migration (ΔruvAB, ΔrecG, ΔradA) activity. RarA contributes to RecA thread formation, that are thought to be the active forms of RecA during homology search. The absence of RarA reduced RecA accumulation, and the formation of visible RecA threads in vivo upon DNA damage. When ΔrarA was combined with mutations in genuine RecA accessory genes, RecA accumulation was further reduced in ΔrarA ΔrecU and ΔrarA ΔrecX double mutant cells, and was blocked in ΔrarA recF15 cells. These results suggest that RarA contributes to the assembly of RecA nucleoprotein filaments onto single-stranded DNA, and possibly antagonizes RecA filament disassembly.

Lymphocyte integration of complement cues.

We address current data, views and puzzles on the emerging topic of regulation of lymphocytes by complement proteins or fragments. Such regulation is believed to take place through complement receptors (CR) and membrane complement regulators (CReg) involved in cell function or protection, respectively, including intracellular signalling. Original observations in B cells clearly support that complement cues through CR improve their performance. Other lymphocytes likely integrate complement-derived signals, as most lymphoid cells constitutively express or regulate CR and CReg upon activation. CR-induced signals, particularly by anaphylatoxins, clearly regulate lymphoid cell function. In contrast, data obtained by CReg crosslinking using antibodies are not always confirmed in human congenital deficiencies or knock-out mice, casting doubts on their physiological relevance. Unsurprisingly, human and mouse complement systems are not completely homologous, adding further complexity to our still fragmentary understanding of complement-lymphocyte interactions.

DNA double strand break end-processing and RecA induce RecN expression levels in Bacillus subtilis.

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.

Early steps of double-strand break repair in Bacillus subtilis.

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.

Detection of the early stage of recombinational DNA repair by silicon nanowire transistors.

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.

RecX facilitates homologous recombination by modulating RecA activities.

The Bacillus subtilis recH342 strain, which decreases interspecies recombination without significantly affecting the frequency of transformation with homogamic DNA, carried a point mutation in the putative recX (yfhG) gene, and the mutation was renamed as recX342. We show that RecX (264 residues long), which shares partial identity with the Proteobacterial RecX (<180 residues), is a genuine recombination protein, and its primary function is to modulate the SOS response and to facilitate RecA-mediated recombinational repair and genetic recombination. RecX-YFP formed discrete foci on the nucleoid, which were coincident in time with RecF, in response to DNA damage, and on the poles and/or the nucleoid upon stochastic induction of programmed natural competence. When DNA was damaged, the RecX foci co-localized with RecA threads that persisted for a longer time in the recX context. The absence of RecX severely impaired natural transformation both with plasmid and chromosomal DNA. We show that RecX suppresses the negative effect exerted by RecA during plasmid transformation, prevents RecA mis-sensing of single-stranded DNA tracts, and modulates DNA strand exchange. RecX, by modulating the "length or packing" of a RecA filament, facilitates the initiation of recombination and increases recombination across species.

Polynucleotide phosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN, SsbA and RecA proteins.

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.

Double-strand break repair in bacteria: a view from Bacillus subtilis.

In all living organisms, the response to double-strand breaks (DSBs) is critical for the maintenance of chromosome integrity. Homologous recombination (HR), which utilizes a homologous template to prime DNA synthesis and to restore genetic information lost at the DNA break site, is a complex multistep response. In Bacillus subtilis, this response can be subdivided into five general acts: (1) recognition of the break site(s) and formation of a repair center (RC), which enables cells to commit to HR; (2) end-processing of the broken end(s) by different avenues to generate a 3'-tailed duplex and RecN-mediated DSB 'coordination'; (3) loading of RecA onto single-strand DNA at the RecN-induced RC and concomitant DNA strand exchange; (4) branch migration and resolution, or dissolution, of the recombination intermediates, and replication restart, followed by (5) disassembly of the recombination apparatus formed at the dynamic RC and segregation of sister chromosomes. When HR is impaired or an intact homologous template is not available, error-prone nonhomologous end-joining directly rejoins the two broken ends by ligation. In this review, we examine the functions that are known to contribute to DNA DSB repair in B. subtilis, and compare their properties with those of other bacterial phyla.

Bacillus subtilis polynucleotide phosphorylase 3'-to-5' DNase activity is involved in DNA repair.

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.

Dynamic structures of Bacillus subtilis RecN-DNA complexes.

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.

High level of conservation in Plasmodium vivax merozoite surface protein 4 (PvMSP4).

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.

MHC class I genes in the owl monkey: mosaic organisation, convergence and loci diversity.

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.