Interaction of branch migration translocases with the Holliday junction-resolving enzyme and their implications in Holliday junction resolution.
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.
From the Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Departamento de Biotecnología Microbiana, 28049 Madrid, Spain and.
Cristina
C
Cañas
Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Yuki
Y
Suzuki
From the Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Departamento de Biotecnología Microbiana, 28049 Madrid, Spain and.
Chiara
C
Marchisone
From the Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Departamento de Biotecnología Microbiana, 28049 Madrid, Spain and.
Begoña
B
Carrasco
From the Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Departamento de Biotecnología Microbiana, 28049 Madrid, Spain and.
Verónica
V
Freire-Benéitez
Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
Kunio
K
Takeyasu
From the Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Departamento de Biotecnología Microbiana, 28049 Madrid, Spain and jcalonso@cnb.cisc.es.
Juan C
JC
Alonso
From the Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Departamento de Biotecnología Microbiana, 28049 Madrid, Spain and sayora@cnb.cisc.es.
Silvia
S
Ayora
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D045566:DNA, Cruciform / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D045566:DNA, Cruciform; C061260:RuvB protein, Bacteria; D005090:Exodeoxyribonucleases; C070765:recJ protein, Bacteria
Atomic Force Microscopy (AFM); DNA Enzyme; DNA Helicase; DNA Repair; DNA-Protein Interaction; Genetic Recombination; Holliday Junction; RecG; RecU; RuvABC