UvrD helicase is required for nucleotide excision repair, although its role in this process is not well defined. Here we show that Escherichia coli UvrD binds RNA polymerase during transcription elongation and, using its helicase/translocase activity, forces RNA polymerase to slide backward along DNA. By inducing backtracking, UvrD exposes DNA lesions shielded by blocked RNA polymerase, allowing nucleotide excision repair enzymes to gain access to sites of damage. Our results establish UvrD as a bona fide transcription elongation factor that contributes to genomic integrity by resolving conflicts between transcription and DNA repair complexes. We further show that the elongation factor NusA cooperates with UvrD in coupling transcription to DNA repair by promoting backtracking and recruiting nucleotide excision repair enzymes to exposed lesions. Because backtracking is a shared feature of all cellular RNA polymerases, we propose that this mechanism enables RNA polymerases to function as global DNA damage scanners in bacteria and eukaryotes.
T cells expressing antigen-specific T-cell receptors (TCRs) can mediate effective tumor regression, but they often also are accompanied by autoimmune responses. To determine the TCR affinity threshold defining the optimal balance between effective antitumor activity and autoimmunity in vivo , we used a unique self-antigen system comprising seven human melanoma gp100(209–217)-specific TCRs spanning physiological affinities (1–100 μM). We found that in vitro and in vivo T-cell responses are determined by TCR affinity, except in one case that was compensated by substantial CD8 involvement. Strikingly, we found that T-cell antitumor activity and autoimmunity are closely coupled but plateau at a defined TCR affinity of 10 µM, likely due to diminished contribution of TCR affinity to avidity above the threshold. Together, these results suggest that a relatively low-affinity threshold is necessary for the immune system to avoid self-damage, given the close relationship between antitumor activity and autoimmunity. The low threshold, in turn, indicates that adoptive T-cell therapy treatment strategies using in vitro-generated high-affinity TCRs do not necessarily improve efficacy.
In bacteria transcription and translation are linked in time and space. When coupled to RNA polymerase (RNAP), the translating ribosome ensures transcriptional processivity by preventing RNAP backtracking. Recent advances in the field have characterized important linker proteins that bridge the gap between transcription and translation: In particular, the NusE(S10):NusG complex and the NusG homolog, RfaH. The direct link between the moving ribosome and RNAP provides a basis for maintaining genomic integrity while enabling efficient transcription and timely translation of various genes within the bacterial cell.
T cell receptors (TCRs) on T cells recognize peptide-major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells and this interaction determines the T cell immune response. Due to negative selection, naturally occurring TCRs bind self (tumor) peptides with low affinity and have a much higher affinity for foreign antigens. This complicates isolation of naturally occurring, high affinity TCRs that mediate more effective tumor rejection for therapeutic purposes. An attractive approach to resolve this issue is to engineer high affinity TCRs in vitro using phage, yeast or mammalian TCR display systems. A caveat of these systems is that they rely on a large library by random mutagenesis due to the lack of knowledge regarding the specific interactions between the TCR and pMHC. We have focused on the mammalian retroviral display system because it uniquely allows for direct comparison of TCR-pMHC-binding properties with T-cell activation outcomes. Through an alanine-scanning approach, we are able to quickly map the key amino acid residues directly involved in TCR-pMHC interactions thereby significantly reducing the library size. Using this method, we demonstrate that for a self-antigen-specific human TCR (R6C12) the key residues for pMHC binding are located in the CDR3β region. This information was used as a basis for designing an efficacious TCR CDR3α library that allowed for selection of TCRs with higher avidity than the wild-type as evaluated through binding and activation experiments. This is a direct approach to target specific TCR residues in TCR library design to efficiently engineer high avidity TCRs that may potentially be used to enhance adoptive immunotherapy treatments.
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