Single-molecule study of RuvAB-mediated Holliday-junction migration

Alexandre Dawid (1) , Vincent Croquette (2) , Mikhail Grigoriev (3) , François Heslot (1)

1 LPA - Laboratoire Pierre Aigrain
2 LPS - Laboratoire de Physique Statistique de l'ENS
3 LBME - Laboratoire de biologie moléculaire eucaryote

Branch migration of Holliday junctions is an important step of genetic recombination and DNA repair. In Escherichia coli, this process is driven by the RuvAB complex acting as a molecular motor. Using magnetic tweezers, we studied the RuvAB-directed migration of individual Holliday junctions formed between two approximate to6-kb DNA molecules of identical sequence, and we measured the migration rate at 37degreesC and 1 mM ATP. We directly demonstrate that RuvAB is a highly processive DNA motor protein that is able to drive continuous and unidirectional branch migration of Holliday junctions at a well defined average speed over several kilobases through homologous sequences. We observed directional inversions of the migration at the DNA molecule boundaries leading to forth-and-back migration of the branch point and allowing us to measure the migration rate in the presence of negative or positive loads. The average migration rate at zero load was found to be approximate to43 bp/sec. Furthermore, the load dependence of the migration rate is small, within the force range of -3.4 pN (hindering force) to +3.4 pN (assisting force).

Mots clés: DNA BRANCH MIGRATION; ESCHERICHIA-COLI RUVA; HOMOLOGOUS RECOMBINATION; CRYSTAL-STRUCTURE; FUNCTIONAL INTERACTIONS; GENETIC-RECOMBINATION; HEXAMERIC RINGS; SUPERCOILED DNA; ATP HYDROLYSIS; IN-VITRO; DNA BRANCH MIGRATION; ESCHERICHIA-COLI RUVA; HOMOLOGOUS RECOMBINATION; CRYSTAL-STRUCTURE; FUNCTIONAL INTERACTIONS; GENETIC-RECOMBINATION; HEXAMERIC RINGS; SUPERCOILED DNA; ATP HYDROLYSIS; IN-VITRO

Domaines: Biochimie, Biologie Moléculaire; Biophysique [physics.bio-ph]

Alexandre Dawid, Vincent Croquette, Mikhail Grigoriev, François Heslot. Single-molecule study of RuvAB-mediated Holliday-junction migration. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101 (32), pp.11611-6. ⟨10.1073/pnas.0404369101⟩. ⟨hal-01239368⟩