Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material

DNA End Resection Controls the Balance between Homologous and Illegitimate Recombination in Escherichia coli

Even a partial loss of function of human RecQ helicase analogs causes adverse effects such as a cancer-prone Werner, Bloom or Rothmund-Thompson syndrome, whereas a complete RecQ deficiency in Escherichia coli is not deleterious for a cell. We show that this puzzling difference is due to different mechanisms of DNA double strand break (DSB) resection in E. coli and humans. Coupled helicase and RecA loading activities of RecBCD enzyme, which is found exclusively in bacteria, are shown to be responsible for channeling recombinogenic 3′ ending tails toward productive, homologous and away from nonproductive, aberrant recombination events. On the other hand, in recB1080/recB1067 mutants, lacking RecBCD’s RecA loading activity while preserving its helicase activity, DSB resection is mechanistically more alike that in eukaryotes (by its uncoupling from a recombinase polymerization step), and remarkably, the role of RecQ also becomes akin of its eukaryotic counterparts in a way of promoting homologous and suppressing illegitimate recombination. The sickly phenotype of recB1080 recQ mutant was further exacerbated by inactivation of an exonuclease I, which degrades the unwound 3′ tail. The respective recB1080 recQ xonA mutant showed poor viability, DNA repair and homologous recombination deficiency, and very increased illegitimate recombination. These findings demonstrate that the metabolism of the 3′ ending overhang is a decisive factor in tuning the balance of homologous and illegitimate recombination in E. coli, thus highlighting the importance of regulating DSB resection for preserving genome integrity. recB mutants used in this study, showing pronounced RecQ helicase and exonuclease I dependence, make up a suitable model system for studying mechanisms of DSB resection in bacteria. Also, these mutants might be useful for investigating functions of the conserved RecQ helicase family members, and congruently serve as a simpler, more defined model system for human oncogenesis

An early history of T cell-mediated cytotoxicity.

After 60 years of intense fundamental research into T cell-mediated cytotoxicity, we have gained a detailed knowledge of the cells involved, specific recognition mechanisms and post-recognition perforin-granzyme-based and FAS-based molecular mechanisms. What could not be anticipated at the outset was how discovery of the mechanisms regulating the activation and function of cytotoxic T cells would lead to new developments in cancer immunotherapy. Given the profound recent interest in therapeutic manipulation of cytotoxic T cell responses, it is an opportune time to look back on the early history of the field. This Timeline describes how the early findings occurred and eventually led to current therapeutic applications

Safety and effectiveness of combination versus monotherapy with immune checkpoint inhibitors in patients with preexisting autoimmune diseases

ABSTRACTPatients with preexisting autoimmune disease (pAID) are generally excluded from clinical trials for immune checkpoint inhibitors (ICIs) for cancer due to concern of flaring pAID. In this multi-center, retrospective observational study, we compared safety of ICI combination (two ICI agents) versus monotherapy in cancer patients with pAIDs. The primary outcome was time to AEs (immune-related adverse events (irAEs) and/or pAID flares), with progression-free survival (PFS) and overall survival as secondary outcomes. Sixty-four of 133 patients (48%) received ICI combination and 69 (52%) monotherapy. Most had melanoma (32%) and lung cancer (31%). Most common pAIDs were rheumatic (28%) and dermatologic (23%). Over a median follow-up of 15 months (95%CI, 11-18 mo), the cumulative incidence of any-grade irAEs was higher for combination compared to monotherapy (subdistribution hazard ratio (sHR) 2.27, 95%CI 1.35–3.82). No statistically significant difference was observed in high-grade irAEs (sHR 2.31 (0.95–5.66), P = .054) or the cumulative incidence of pAID flares. There was no statistically significant difference for melanoma PFS between combination versus monotherapy (23.2 vs. 17.1mo, P = .53). The combination group was more likely to discontinue or hold ICI, but > 50% of the combination group was still able to continue ICI therapy. No treatment-related deaths occurred. In our cohort with pAIDs, patients had a tolerable toxicity profile with ICI combination therapy. Our results support the use of ICI combination if deemed necessary for cancer therapy in patients with pAIDs, since the ICI toxicities were comparable to monotherapy, able to be effectively managed and mostly did not require ICI interruption