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Originally published as Genetics Published Articles Ahead of Print on July 14, 2005.

Genetics, Vol. 171, 873-883, November 2005, Copyright © 2005
doi:10.1534/genetics.105.045906

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The RuvAB Branch Migration Translocase and RecU Holliday Junction Resolvase Are Required for Double-Stranded DNA Break Repair in Bacillus subtilis

Humberto Sanchez*, Dawit Kidane{dagger},1, Patricia Reed{ddagger}, Fiona A. Curtis{ddagger}, M. Castillo Cozar*, Peter L. Graumann{dagger},1, Gary J. Sharples{ddagger} and Juan C. Alonso*,2

* Department of Microbial Biotechnology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain, {dagger} Biochemie, Fachbereich Chemie, Philipps-Universität Marburg, 35032 Marburg, Germany and {ddagger} Centre for Infectious Diseases, Wolfson Research Institute, University of Durham, Stockton-on-Tees TS17 6BH, United Kingdom

2 Corresponding author: Departmento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, C/Darwin 3, Cantoblanco, 28049 Madrid, Spain.
E-mail: jcalonso{at}cnb.uam.es

In models of Escherichia coli recombination and DNA repair, the RuvABC complex directs the branch migration and resolution of Holliday junction DNA. To probe the validity of the E. coli paradigm, we examined the impact of mutations in {Delta}ruvAB and {Delta}recU (a ruvC functional analog) on DNA repair. Under standard transformation conditions we failed to construct {Delta}ruvAB {Delta}recG, {Delta}recU {Delta}ruvAB, {Delta}recU {Delta}recG, or {Delta}recU {Delta}recJ strains. However, {Delta}ruvAB could be combined with addAB (recBCD), recF, recH, {Delta}recS, {Delta}recQ, and {Delta}recJ mutations. The {Delta}ruvAB and {Delta}recU mutations rendered cells extremely sensitive to DNA-damaging agents, although less sensitive than a {Delta}recA strain. When damaged cells were analyzed, we found that RecU was recruited to defined double-stranded DNA breaks (DSBs) and colocalized with RecN. RecU localized to these centers at a later time point during DSB repair, and formation was dependent on RuvAB. In addition, expression of RecU in an E. coli ruvC mutant restored full resistance to UV light only when the ruvAB genes were present. The results demonstrate that, as with E. coli RuvABC, RuvAB targets RecU to recombination intermediates and that all three proteins are required for repair of DSBs arising from lesions in chromosomal DNA.




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