Interaction of Branch Migration Translocases with the Holliday Junction-resolving Enzyme and Their Implications in Holliday Junction Resolution

Cristina Cañas,Yuki Suzuki,Chiara Marchisone,Begoña Carrasco,Verónica Freire-Benéitez,Kunio Takeyasu,Juan C Alonso,Silvia Ayora

doi:10.1074/jbc.m114.552794

Abstract

Double-strand break repair involves the formation of Holliday junction (HJ) structures that need to be resolved to promote correct replication and chromosomal segregation. The molecular mechanisms of HJ branch migration and/or resolution are poorly characterized in Firmicutes. Genetic evidence suggested that the absence of the RuvAB branch migration translocase and the RecU HJ resolvase is synthetically lethal in Bacillus subtilis, whereas a recU recG mutant was viable. In vitro RecU, which is restricted to bacteria of the Firmicutes phylum, binds HJs with high affinity. In this work we found that RecU does not bind simultaneously with RecG to a HJ. RuvB by interacting with RecU bound to the central region of HJ DNA, loses its nonspecific association with DNA, and re-localizes with RecU to form a ternary complex. RecU cannot stimulate the ATPase or branch migration activity of RuvB. The presence of RuvB·ATPγS greatly stimulates RecU-mediated HJ resolution, but the addition of ATP or RuvA abolishes this stimulatory effect. A RecU·HJ·RuvAB complex might be formed. RecU does not increase the RuvAB activities but slightly inhibits them.

Highlights

Bacillus subtilis RuvAB and RecG translocases and RecU resolvase are crucial in homologous recombination
The binding of RuvB to Holliday junction (HJ) in the presence of RecU was analyzed under conditions that neither allowed RecU HJ cleavage nor RuvB HJ branch migration and that were previously used (e.g. 15 mM CaCl2 and 1 mM ATP; see Ref 44)
A RuvB1⁄7HJ complex was not detected in the presence of 5 mM MgCl2 and 1 mM ATP␥S even if 0.1% glutaraldehyde was added before electrophoresis

Summary

Background

Bacillus subtilis RuvAB and RecG translocases and RecU resolvase are crucial in homologous recombination. Genetic studies suggested that RecU could work independently of RuvAB and in coordination with the RecG helicase This is consistent with the observations that: (i) the ruvA, ruvB, recU, or recG mutation renders cells very sensitive to DNA-damaging agents [24, 25]; (ii) RuvA, RecU, and RecG share common suppressors [32]; (iii) ⌬ruvAB ⌬recG and ⌬ruvAB ⌬recU strains are synthetically lethal, whereas a ⌬recG ⌬recU strain could be obtained, with reduced viability [24, 25]; (iv) the ruvA and ruvB genes are SOS inducible, whereas the recU gene is located in another operon under the control of ␴M, and its expression is increased upon cell envelop stress (e.g. vancomycin addition or by acid, heat, ethanol, and superoxide stresses) [33, 34]. These results altogether prompted us to analyze the possible coordination of RecU with the RuvAB and RecG translocases

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION