Location of Phosphorylation Sites within Long Polypeptide Chains by Binder-Assisted Nanopore Detection.

The detection and mapping of protein phosphorylation sites are essential for understanding the mechanisms of various cellular processes and for identifying targets for drug development. The study of biopolymers at the single-molecule level has been revolutionized by nanopore technology. In this study, we detect protein phosphorylation within long polypeptides (>700 amino acids), after the attachment of binders that interact with phosphate monoesters; electro-osmosis is used to drive the tagged chains through engineered protein nanopores.

Enzyme-less nanopore detection of post-translational modifications within long polypeptides.

Means to analyse cellular proteins and their millions of variants at the single-molecule level would uncover substantial information previously unknown to biology. Nanopore technology, which underpins long-read DNA and RNA sequencing, holds potential for full-length proteoform identification. We use electro-osmosis in an engineered charge-selective nanopore for the non-enzymatic capture, unfolding and translocation of individual polypeptides of more than 1,200 residues.

Single-Molecule Tethered Particle Motion Studies on the DNA Recombinase Filament Assembly and Disassembly.

Bacterial RecA and eukaryotic Rad51 are recombinases indispensable for DNA homologous recombination and repair of double-stranded DNA breaks. Understanding the functions and biophysical properties of the DNA recombinases benefits the research in human medicine such as cancer biology. Single-molecule techniques provide the mechanistic details of complex biological reactions.

Rad51 tolerates mismatches in hybrid meiosis with diverse genome sequences.

Most eukaryotes possess two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1) to promote interhomolog recombination during meiosis. However, some eukaryotes have lost Dmc1. Given that mammalian and yeast () Dmc1 have been shown to stabilize recombination intermediates containing mismatches better than Rad51, we used the Pezizomycotina filamentous fungus to address if and how Rad51-only eukaryotes conduct interhomolog recombination in zygotes with high sequence heterogeneity.

Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures.

Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open "triplet gate" for mismatch tolerance.

Rad51 facilitates filament assembly of meiosis-specific Dmc1 recombinase.

Dmc1 recombinases are essential to homologous recombination in meiosis. Here, we studied the kinetics of the nucleoprotein filament assembly of Dmc1 using single-molecule tethered particle motion experiments and in vitro biochemical assay. ScDmc1 nucleoprotein filaments are less stable than the ScRad51 ones because of the kinetically much reduced nucleation step.