Biology subjects

Berger, J. M.

Publications and source records attributed to Berger, J. M..

22 records · Page 2Linked to original sources

bioRxiv2020

The molecular coupling between substrate recognition and ATP turnover in a AAA+ hexameric helicase loader

In many bacteria and in eukaryotes, replication fork establishment requires the controlled loading of hexameric, ring-shaped helicases around DNA by AAA+ ATPases. How loading factors use ATP to control helicase deposition is poorly understood. Here, we dissect how specific ATPase elements of E. coli DnaC, an archetypal loader for the bacterial DnaB helicase, play distinct roles in helicase loading and the activation of DNA unwinding. We identify a new element, the arginine-coupler, which regulates the switch-like behavior of DnaC to prevent futile ATPase cycling and maintains loader responsiveness to replication restart systems. Our data help explain how the ATPase cycle of a AAA+-family helicase loader is channeled into productive action on its target; comparative studies indicate elements analogous to the Arg-coupler are present in related, switch-like AAA+ proteins that control replicative helicase loading in eukaryotes, as well as polymerase clamp loading and certain classes of DNA transposases.

biochemistry↗

bioRxiv2020

Structural and functional characterization of the Spo11 core complex

Spo11, which makes DNA double-strand breaks (DSBs) essential for meiotic recombination, is poorly understood mechanistically because it has been recalcitrant to biochemical study. Here, we provide a molecular analysis of S. cerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal Topoisomerase VI, with Rec104 similar to a GHKL domain but without conserved ATPase motifs. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), indicating that dimerization may control DSB formation. Reconstitution of DNA binding reveals topoisomerase-like preferences for duplex-duplex junctions and bent DNA. Spo11 also binds noncovalently but with high affinity to DNA ends mimicking cleavage products, suggesting a mechanism to cap DSB ends. Mutations that reduce DNA binding in vitro attenuate DSB formation, alter DSB processing, and reshape the DSB landscape in vivo. Our data reveal structural and functional similarities between the Spo11 core complex and Topo VI, but also highlight differences reflecting their distinct biological roles.

molecular biology↗

bioRxiv2019

Toward computational design of protein crystals with improved resolution

Substantial advances have been made in the computational design of protein interfaces over the last 20 years. However, the interfaces targeted by design have typically been stable and high affinity. Here, we report the development of a generic computational design method to stabilize the weak interactions at crystallographic interfaces. Initially, we analyzed structures reported in the Protein Data Bank (PDB) to determine whether crystals with more stable interfaces result in higher resolution structures. We found that, for twenty-two variants of a single protein crystallized by a single individual, Rosetta score correlates with resolution. We next developed and tested a computational design protocol, seeking to identify point mutations that would improve resolution on a highly stable variant of staphylococcal nuclease (SNase {Delta}+PHS). Only one of eleven initial designs crystallized, forcing us to re-evaluate our strategy and base our designs on an ensemble of protein backbones. Using this strategy, four of the five designed proteins crystallized. Collecting diffraction data for multiple crystals per design and solving crystal structures, we found that designed crystals improved resolution modestly and in unpredictable ways, including altering crystal space group. Post-hoc, in silico analysis showed that crystal space groups could have been predicted for four of six variants (including WT), but that resolution did not correlate with interface stability, as it did in the preliminary results. Our results show that single point mutations can have significant effects on crystal resolution and space group, and that it is possible to computationally identify such mutations, suggesting a potential design strategy to generate high-resolution protein crystals from poorly diffracting ones.

biophysics↗

bioRxiv2018

A Primase-Induced Conformational Switch Controls the Stability of the Bacterial Replisome

Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly-found plasticity lies in the >400-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.

biochemistry↗