Biology subjects

Butryn, A.

Publications and source records attributed to Butryn, A..

4 recordsLinked to original sources

bioRxiv2026

Structure of the Pre-Initiation Complex Explains CMGE Biogenesis

Summary paragraphWhen cells enter S phase, bidirectional DNA replication is initiated through the kinase-regulated recruitment of three activators (Cdc45, GINS and Pol epsilon) to a duplex DNA-loaded double hexamer of MCM ATPases. Together these proteins form two CMGE helicases that establish divergent replication forks as they become separated1. To understand CMGE biogenesis, we reconstituted the pre-Initiation Complex with purified yeast proteins. The cryo-EM structure shows a set of firing factors caught in the act of assembling two symmetric CMGEs. We show how stepwise complex formation reshapes MCM in preparation for DNA opening and we explain how ATP promotes firing-factor ejection and CMGE maturation. While we find that Sld2 promotes GINS recruitment to MCM as expected, it also aids efficient separation of the CMGE dimer, and it is essential for lagging strand ejection from MCM. These findings have direct implications for our understanding of the metazoan Sld2 ortholog, RECQL4, pointing to a replication-fork establishment mechanism conserved across eukaryotes.

biochemistry↗

bioRxiv2026

Mcm10 braces converging replicative helicases to pull apart origin DNA

Eukaryotic DNA replication initiation requires controlled, temporally separated steps to preserve genome stability. The MCM replicative helicase is loaded on duplex DNA as an inactive double hexamer, which nucleates the assembly of dimeric Cdc45-MCM-GINS-Pol epsilon (dCMGE) replisomes. Mcm10 splits dCMGE into two, generating divergent replication forks, but the mechanism is unknown. Using ATPase-defective yeast MCM variants that slow origin melting, we captured five reaction intermediates that explain the structural mechanism. Two Mcm10 molecules bridge across the CMGE dimer, bracing two converging helicases. The restrained MCM motors pull apart the two DNA filaments, such that each lagging strand becomes ejected through the Mcm2-5 gate. Our reconstituted structures resemble the double CMG stabilized by metazoan DONSON, pointing to an origin DNA melting mechanism conserved across evolution.

biochemistry↗

bioRxiv2025

Time-resolved ligand dynamics revealed in a β-lactamase using room-temperature serial crystallography

{beta}-Lactamases catalyze {beta}-lactam antibiotic hydrolysis and are important contributors to bacterial antimicrobial resistance; {beta}-lactamase inhibitors are widely used to overcome {beta}-lactamase-mediated antibiotic resistance. Nucleophilic serine {beta}-lactamases (SBLs) react with their substrates and clinically available inhibitors via a covalent reaction to give complexes which can undergo further reaction. Using room temperature drop on fixed target serial crystallography, where ligands are rapidly mixed with microcrystals, and classical single-crystal crystallography at cryogenic temperatures, we investigate the reversible covalent reaction of the SBL CTX-M-15 with the diazobicyclooctane inhibitor avibactam. We observe avibactam covalently reacted (ring-opened) with the nucleophilic Ser70, at timepoints from 80 ms to minutes (room temperature) and hours (100 K). These crystallographic data reveal time-dependent movement of the avibactam carbamoyl complex, from 1.3 s onwards, that has implications for the 5-exo-trig recyclization mechanism that determines inhibitor reformation. Combined with molecular dynamics simulations and quantum mechanics calculations at the density functional theory level, the results show that in the first seconds of the reaction the avibactam N-sulfate nitrogen is poorly positioned for recyclization. This subsequently equilibrates after 10 s to a stable endpoint that is in a conformation potentially primed to initiate recyclization through attack of the N-sulfate nitrogen on the carbamoyl carbon. These results further demonstrate the capacity of room-temperature serial crystallography to capture time-resolved changes in ligand conformation at an enzyme active site, complementing discrete classical cryo-crystallography. These data inform on ligand dynamics and the stereoelectronics of diazobicyclooctane inhibition, aiding drug discovery efforts to develop inhibitors of nucleophilic enzymes.

biochemistry↗

bioRxiv2022

Redox-controlled structural reorganization and flavin strain within the ribonucleotide reductase R2b-NrdI complex monitored by serial femtosecond crystallography

Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b-NrdI complex, a di-metal carboxylate- flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free-electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. We show that the flavin in the hydroquinone state is under steric strain in the R2b-NrdI protein complex, presumably tuning its redox potential to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2-interacton site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b- NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.

biochemistry↗