Biology subjects

Tolun, G.

Publications and source records attributed to Tolun, G..

3 recordsLinked to original sources

bioRxiv2026

Structural basis of the nucleotide incorporation cycle of bacterial DNA polymerase III

Bacterial DNA polymerase III (PolIII) is the primary replicative polymerase responsible for faithful genome duplication, yet high-resolution structural snapshots of the enzyme during DNA synthesis and proofreading have remained elusive. Here we determine cryo-EM structures of the Escherichia coli PolIII core-clamp-DNA binary, ternary and proofreading complexes at 2.4-2.7 [A] resolution, revealing the structural basis of nucleotide selection, incorporation and proofreading. Binding of an incoming nucleotide expels the single-stranded template overhang from the polymerase central channel and induces a large rotation of the polymerase index finger, which closes around the incoming nucleotide to form a compact nascent base-pair-binding pocket. A conserved tyrosine on the finger stacks against the incoming nucleotide to impose stringent steric constraints that promote nucleotide selection, whereas a conserved histidine acts as a steric gate to exclude ribonucleotides. The proofreading structures capture key intermediates that reveal how PolIII transfers a mismatched primer terminus to the 3'-5' exonuclease active site and excises the misincorporated nucleotide while avoiding unnecessary degradation of correctly paired DNA. Together, these findings define the molecular mechanism by which the bacterial replicative polymerase couples rapid and processive DNA synthesis with exceptional replication fidelity.

biochemistry↗

bioRxiv2023

Structural characterisation of the complete cycle of sliding clamp loading in Escherichia coli

Ring-shaped DNA sliding clamps are essential for DNA replication and genome maintenance. Clamps need to be opened or trapped open and chaperoned onto DNA by clamp loader complexes (CLCs). Detailed understanding of the mechanisms by which CLCs open and place clamps around DNA remains limited. Here, we present a series of six structures of the Escherichia coli CLC bound to an open or closed clamp on and off a primer-template DNA that represent all intermediates in the clamp loading process. We show that the ATP-bound CLC first binds to a clamp, then constricts to hold onto it. The CLC then expands to open the clamp with a gap large enough for double-stranded DNA to enter. Upon binding to DNA, the CLC constricts slightly, allowing ATP hydrolysis and clamp closing around DNA. Although both yeast and E. coli CLCs open clamps by crab claw-like motions, they do it by the CLC expanding in opposite directions. These structures provide critical high-resolution snapshots of clamp loading by the E. coli CLC, revealing how the molecular machine works.

biochemistry↗

bioRxiv2022

Structure of phage lambda Red-beta(177) annealase shows how it anneals DNA strands during single-strand annealing homologous DNA recombination

The bacteriophage {lambda} red recombination system catalyzes the single-strand annealing homologous DNA recombination reaction, in which Red{beta} annealase protein plays a critical role. Using cryogenic electron microscopy, we were able to determine a structure of a C-terminally truncated Red{beta} with the residues 1-177 bound to two complementary 27mer oligonucleotides forming an annealing intermediate, to a final resolution of 3.3 [A]. This structure validates and rationalizes decades of experimental observations on the biochemistry of Red{beta}. Definition of the interaction surfaces between subunits explains not only the DNA binding properties of Red{beta}, but also its propensity to oligomerize into long helical filaments, which are also formed by almost all annealases and are known to be functionally important. In addition, this annealing intermediate structure provides a detailed picture of the hydrogen bonding network that positions the DNA strands in a planar orientation to facilitate base pairing. Residues 133-138, which are missing from our structure, form a flexible loop. Molecular dynamics simulations were used to model the range of motion of the flexible loop, which suggested that it has a crucial role in keeping the DNA strands in the DNA-binding groove long enough to allow homology searching. The combination of structural and computational observations has allowed us to propose a detailed mechanism for the action of Red{beta}. More than half a century after its discovery, our work shines a light not only on the structure and mechanisms of Red{beta}, but also of other proteins within the annealase superfamilies. Significance StatementSingle-strand annealing homologous DNA recombination is a process that is conserved throughout evolution from bacteriophages to humans, highlighting its importance and indispensability. It is a process that repairs double-stranded DNA breaks and is particularly vital in dsDNA viruses. The Red{beta} protein from the bacteriophage lambda is the archetypal annealase protein, forming the basis of our knowledge about this class of proteins. Along with the exonuclease {lambda}Exo, these two proteins not only form the model system for single-strand annealing homologous recombination, but are also used in thousands of laboratories worldwide for performing genetic manipulations. After its discovery in 1966, we report the first structure of the DNA-binding and oligomerization domain of Red{beta}, providing details about the mechanism of homologous DNA annealing.

biochemistry↗