Biology subjects

Strick, T. R.

Publications and source records attributed to Strick, T. R..

3 recordsLinked to original sources

bioRxiv2024

Competition between glycine and GABAA receptors for gephyrin controls their equilibrium populations at inhibitory synapses

Glycine and GABA receptors are ligand-gated chloride channels that mediate inhibitory neurotransmission throughout the central nervous system. The receptors co-localise widely at inhibitory synapses in the spinal cord and in the brainstem due to their interaction with an overlapping binding site of the synaptic scaffold protein gephyrin, pointing to a direct competition between the different receptor types. We have put this hypothesis to the test using single molecule approaches to measure receptor-gephyrin interactions in cells and in vitro. We explored the effects of receptor competition at inhibitory synapses in living neurons by measuring the change in the accumulation and effective stabilisation energy of glycine receptors in the presence of interfering GABA receptor complexes through single molecule tracking and diffusion analysis. Secondly, using molecular tweezers, we quantified the thermodynamic properties of receptor-gephyrin binding, demonstrating direct and reversible competition through the addition of interacting peptides in solution. The relatively low affinity of GABA receptor subunits for gephyrin compared to the glycine receptor raises interesting questions about the role of this competition in synaptic plasticity. We hypothesize that GABA and glycine receptor competition constitutes a molecular system designed to reconcile synapse stability and plasticity at mixed inhibitory synapses.

neuroscience↗

bioRxiv2022

Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation

Streptococcus pyogenes CRISPR-Cas9 (SpCas9) stabilizes each strand of a DNA bubble via distinct interactions, forming a stable ~20 bp R-loop on the complementary strand but relying on narrower protein-DNA interactions to bind the non-complementary strand with 5 NGG protospacer adjacent motif (PAM). The enzymes HNH endonuclease domain cleaves DNA within the R-loop, and its RuvC endonuclease cleaves 5 to the NGG and opposite the HNH cleavage site to generate blunt DNA ends. We show that this nucleoprotein complex can be mechanically strained by shifting the RNA:DNA hybrid and that the HNH cleavage site tracks this shift but the RuvC cleavage site does not, resulting in overhanging DNA ends. This is observed using cleavage in living cells and sequencing assays to characterize overhangs generated by stressed complexes, as well as single-molecule cleavage assays to characterize differential cleavage kinetics of HNH and RuvC endonucleases in stressed complexes as well as complex disassembly. Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the SpCas9 complex responds to internal mechanical strain by robustly generating overhanging, rather than blunt, DNA ends. Internal mechanical strain is generated by increasing or decreasing the spacing between the RNA:DNA hybrid and the downstream canonical PAM. Up to two-base 3 overhangs can be robustly generated via a two-base increase in the distance between hybrid and PAM. We also use single-molecule experiments to reconstruct the full course of the CRISPR-SpCas9 reaction in real-time, monitoring and quantifying the R-loop formation, the first and second DNA incision events, and the post-catalytic complex dissociation. Complex dissociation and release of broken DNA ends appears to be a rate-limiting step of the reaction, and strained SpCas9 are sufficiently destabilized so as to rapidly dissociate after formation of broken DNA ends.

genomics↗

bioRxiv2018

The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.

biochemistry↗