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Published 1 August 2005. doi:10.1083/jcb.200412090
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 170, Number 3, 357-366
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Article

 Dynamic formation of RecA filaments at DNA double strand break repair centers in live cells

Dawit Kidane and Peter L. Graumann

Biochemie, Fachbereich Chemie, Hans-Meerwein-Straße, Philipps-Universität Marburg, 35032 Marburg, Germany

Correspondence to Peter L. Graumann: graumann{at}staff.uni-marburg.de

We show that RecN protein is recruited to a defined DNA double strand break (DSB) in Bacillus subtilis cells at an early time point during repair. Because RecO and RecF are successively recruited to DSBs, it is now clear that dynamic DSB repair centers (RCs) exist in prokaryotes. RecA protein was also recruited to RCs and formed highly dynamic filamentous structures, which we term threads, across the nucleoids. Formation of RecA threads commenced ~30 min after the induction of DSBs, after RecN recruitment to RCs, and disassembled after 2 h. Time-lapse microscopy showed that the threads rapidly changed in length, shape, and orientation within minutes and can extend at 1.02 µm/min. The formation of RecA threads was abolished in recJ addAB mutant cells but not in each of the single mutants, suggesting that RecA filaments can be initiated via two pathways. Contrary to proteins forming RCs, DNA polymerase I did not form foci but was present throughout the nucleoids (even after induction of DSBs or after UV irradiation), suggesting that it continuously scans the chromosome for DNA lesions.

D. Kidane's and P.L. Graumann's present address is Institut für Mikrobiologie, Albert-Ludwigs Universität Freiburg, 79104 Freiburg, Germany.

Abbreviations used in this paper: Cm, chloramphenicol; DSB, double strand break; HO, homothallic; MMC, mitomycin C; Pol, polymerase; RC, repair center; spec, spectinomycin; ss, single stranded; tet, tetracycline.


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