The UvrABC endonuclease from Escherichia coli repairs damage in the DNA by dual incision of the damaged strand on both sides of the lesion. The incisions are in an ordered fashion, first on the 3-side and next on the 5-side of the damage, and they are the result of binding of UvrC to the UvrB-DNA preincision complex. In this paper, we show that at least the C-terminal 24 amino acids of UvrB are involved in interaction with UvrC and that this binding is important for the 3-incision. The C-terminal region of UvrB, which shows homology with a domain of the UvrC protein, is part of a region that is predicted to be able to form a coiled-coil. We therefore propose that UvrB and UvrC interact through the formation of such a structure. The C-terminal region of UvrB only interacts with UvrC when present in the preincision complex, indicating that the conformational change in UvrB accompanying the formation of this complex exposes the UvrC binding domain. Binding of UvrC to the C-terminal region of UvrB is not important for the 5-incision, suggesting that for this incision a different interaction of UvrC with the UvrB-DNA complex is required. Truncated UvrB mutants that lack up to 99 amino acids from the C terminus still give rise to significant incision (1-2%), indicating that this C-terminal region of UvrB does not participate in the formation of the active site for 3-incision. This region, however, contains the residue (Glu-640) that was proposed to be involved in 3-catalysis, since a mutation of the residue (E640A) fails to promote 3-incision (Lin, J. J., Phillips, A. M., Hearst, J. E., and Sancar, A. (1992) J. Biol. Chem. 267, 17693-17700). We have isolated a mutant UvrB with the same E640A substitution, but this protein shows normal UvrC binding and incision in vitro and also results in normal survival after UV irradiation in vivo. As a consequence of these results, it is still an open question as to whether the catalytic site for 3-incision is located in UvrB, in UvrC, or is formed by both proteins.The UvrB protein is a key subunit of the UvrABC endonuclease from Escherichia coli involved in the repair of a wide range of different types of DNA damage (for reviews, see Refs. 1-3). Together with UvrA, UvrB forms a trimeric UvrA 2 B complex, which is able to recognize a lesion in the DNA. Upon binding to the lesion, the helicase activity of the UvrA 2 B complex results in the loading of the UvrB on the DNA (4) and the subsequent release of the UvrA protein (5). The DNA in the resulting UvrB-DNA preincision complex is unwound (6) and kinked (7), and the UvrB protein seems to be bound on the opposite side of the DNA helix to the site of the damage (8). Upon binding of UvrC to the preincision complex, the DNA is incised on both sides of the lesion. First, the 3Ј-incision is made (9) at the 4th or 5th phosphodiester bond downstream from the lesion, followed by the 5Ј-incision at the 8th phosphodiester bond upstream from the lesion (10, 11). When UvrA is still present in the preincision complex, this can lead to an additio...

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Antigen-specific, MHC-restricted αβ T cells are necessary for protective immunity against Mycobacterium tuberculosis, but the ability to broadly study these responses has been limited. In the present study, we used single-cell and bulk T cell receptor (TCR) sequencing and the GLIPH2 algorithm to analyze M. tuberculosis-specific sequences in two longitudinal cohorts, comprising 166 individuals with M. tuberculosis infection who progressed to either tuberculosis (n = 48) or controlled infection (n = 118). We found 24 T cell groups with similar TCR-β sequences, predicted by GLIPH2 to have common TCR specificities, which were associated with control of infection (n = 17), and others that were associated with progression to disease (n = 7). Using a genome-wide M. tuberculosis antigen screen, we identified peptides targeted by T cell similarity groups enriched either in controllers or in progressors. We propose that antigens recognized by T cell similarity groups associated with control of infection can be considered as high-priority targets for future vaccine development.

Understanding the mechanisms and impact of booster vaccinations are essential in the design and delivery of vaccination programs. Here we show that a three dose regimen of a synthetic peptide vaccine elicits an accruing CD8+ T cell response against one SARS-CoV-2 Spike epitope. We see protection against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, but two dose approaches are insufficient to confer protection. The third vaccine dose of the single T cell epitope peptide results in superior generation of effector-memory T cells and tissue-resident memory T cells, and these tertiary vaccine-specific CD8+ T cells are characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping shows that a substantial fraction of the tertiary CD8+ effector-memory T cells develop from re-migrated tissue-resident memory T cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8+ T cell response leading to protection against otherwise lethal SARS-CoV-2 infection.

Cytotoxic CD8+ T cells mediate cellular immunity through recognition of specific antigens presented by MHC class I on all nucleated cells. Studying T cell interactions and responses provides invaluable information on infection, autoimmunity and cancer. Fluorescently labeled multimers of MHC I can be used to quantify, characterize, and isolate specific CD8+ T cells by flow cytometry. Here we describe the production and use of conditional MHC I multimers that can be loaded with peptides of choice by incubating them at a defined temperature. Multimers are folded with a template peptide that forms a stable complex at low temperature, but dissociates at a defined elevated temperature. Using this technology multiple MHC I multimers can be generated in parallel, to allow staining and isolation of large sets of antigen‐specific CD8+ T cells, especially when combined with barcoding technologies. © 2019 The Authors.