Biology subjects

Washington, M. T.

Publications and source records attributed to Washington, M. T..

2 recordsLinked to original sources

bioRxiv2021

KERA: Analysis Tool for Multi-Process, Multi-State Single-Molecule Data

Molecular machines within cells dynamically assemble, disassemble, and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy (CoSMoS), in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy (TIRFM) approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell-time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERAs utility by analyzing conformational dynamics of two DNA binding proteins: RPA and XPD helicase.

biophysics↗

bioRxiv2020

Visualizing Rev1 Catalyze Protein-template DNA Synthesis

During DNA replication, replicative DNA polymerases may encounter DNA lesions, which can stall replication forks. One way to prevent replication fork stalling is through the recruitment of specialized translesion synthesis (TLS) polymerases that have evolved to incorporate nucleotides opposite DNA lesions. Rev1 is a specialized TLS polymerase that bypasses abasic sites as well as minor-groove and exocyclic guanine adducts. It does this by using a unique protein-template mechanism in which the template base is flipped out of the DNA helix and the incoming dCTP hydrogen bonds with an arginine side chain. To observe Rev1 catalysis at the atomic level, we employed time-lapse X-ray crystallography. We found that Rev1 flips out the template base prior to binding the incoming nucleotide. Binding the incoming nucleotide changes the conformation of the DNA substrate to orient it for nucleotidyl transfer, and this is not coupled to large structural changes in the protein like those observed with other DNA polymerases. Moreover, we found that following nucleotide incorporation, Rev1 converts the pyrophosphate product to two mono-phosphates, which drives the reaction in the forward direction. Following nucleotide incorporation, the hydrogen bonds between the incorporated nucleotide and the arginine side chain are broken, but the templating base remains extrahelical. These post-catalytic changes prevent potentially mutagenic processive synthesis by Rev1 and facilitate dissociation of the DNA product from the enzyme.

biochemistry↗