Mechanism of AAA+ ATPase-mediated RuvAB-Holliday junction branch migration

TY - JOUR

T1 - Mechanism of AAA+ ATPase-mediated RuvAB-Holliday junction branch migration

AU - Wald, Jiri

AU - Fahrenkamp, Dirk

AU - Goessweiner-Mohr, Nikolaus

AU - Lugmayr, Wolfgang

AU - Ciccarelli, Luciano

AU - Vesper, Oliver

AU - Marlovits, Thomas C

N1 - © 2022. The Author(s).

PY - 2022/9

Y1 - 2022/9

N2 - The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life1. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, which assemble together with the RuvA-Holliday junction complex to energize the strand-exchange reaction2. Despite its importance for chromosome maintenance, the structure and mechanism by which this complex facilitates branch migration are unknown. Here, using time-resolved cryo-electron microscopy, we obtained structures of the ATP-hydrolysing RuvAB complex in seven distinct conformational states, captured during assembly and processing of a Holliday junction. Five structures together resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange and context-specific conformational changes in RuvB. Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. Immobilization of the converter enables RuvB to convert the ATP-contained energy into a lever motion, which generates the pulling force driving the branch migration. We show that RuvB motors rotate together with the DNA substrate, which, together with a progressing nucleotide cycle, forms the mechanistic basis for DNA recombination by continuous branch migration. Together, our data decipher the molecular principles of homologous recombination by the RuvAB complex, elucidate discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors and provide a blueprint for the design of state-specific compounds targeting AAA+ motors.

AB - The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life1. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, which assemble together with the RuvA-Holliday junction complex to energize the strand-exchange reaction2. Despite its importance for chromosome maintenance, the structure and mechanism by which this complex facilitates branch migration are unknown. Here, using time-resolved cryo-electron microscopy, we obtained structures of the ATP-hydrolysing RuvAB complex in seven distinct conformational states, captured during assembly and processing of a Holliday junction. Five structures together resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange and context-specific conformational changes in RuvB. Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. Immobilization of the converter enables RuvB to convert the ATP-contained energy into a lever motion, which generates the pulling force driving the branch migration. We show that RuvB motors rotate together with the DNA substrate, which, together with a progressing nucleotide cycle, forms the mechanistic basis for DNA recombination by continuous branch migration. Together, our data decipher the molecular principles of homologous recombination by the RuvAB complex, elucidate discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors and provide a blueprint for the design of state-specific compounds targeting AAA+ motors.

KW - ATPases Associated with Diverse Cellular Activities/genetics

KW - Adenosine Triphosphate

KW - Bacterial Proteins/metabolism

KW - Cryoelectron Microscopy

KW - DNA

KW - DNA Helicases/genetics

KW - DNA, Cruciform

KW - DNA-Binding Proteins/metabolism

KW - Escherichia coli/genetics

KW - Escherichia coli Proteins/genetics

U2 - 10.1038/s41586-022-05121-1

DO - 10.1038/s41586-022-05121-1

M3 - SCORING: Journal article

C2 - 36002576

VL - 609

SP - 630

EP - 639

JO - NATURE

JF - NATURE

SN - 0028-0836

IS - 7927

ER -