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Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre

Nature Cell Biology volume 5, pages 572–577 (2003)Cite this article

Abstract

DNA double-strand break repair (DSBR) is an essential process for preserving genomic integrity in all organisms. To investigate this process at the cellular level, we engineered a system of fluorescently marked DNA double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae to visualize in vivo DSBR in single cells. Using this system, we demonstrate for the first time that Rad52 DNA repair foci and DSBs colocalize. Time-lapse microscopy reveals that the relocalization of Rad52 protein into a focal assembly is a rapid and reversible process. In addition, analysis of DNA damage checkpoint-deficient cells provides direct evidence for coordination between DNA repair and subsequent release from checkpoint arrest. Finally, analyses of cells experiencing multiple DSBs demonstrate that Rad52 foci are centres of DNA repair capable of simultaneously recruiting more than one DSB.

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Figure 1: Localization of a Rad52 focus to a specific DSB.
Figure 2: Rapid assembly and disassembly of spontaneous Rad52 foci.
Figure 3: Repair of multiple DNA DSBs.
Figure 4: Localization of two fluorescently marked DSBs.

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Acknowledgements

The red- and blue-shifted enhanced variants (YFP(10C) and CFP(W7)) of the GFP gene and the DNA sequence encoding the monomeric version of DsRed (mRFP1) were generous gifts from R. Tsien (University of California, San Diego, CA). pJH132 was kindly provided by J.Haber (Brandeis University, Waltham, MA). pCAGS1 was a gift from M. Jasin (Memorial Sloan-Kettering Cancer Center, New York, NY). We also acknowledge the gift of yeast strains from K. Nasmyth (Research Institute of Molecular Pathology, Vienna, Austria) and A. Murray (Harvard University, Cambridge, MA). Finally, we thank J. Hudson for his generous gift of the microscope and T. Tonnessen for research support. M. L. was supported by a fellowship from the Danish Natural Science Research Council. This research was also supported by grants from the NIH to R. R.

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Authors and Affiliations

  1. Department of Genetics & Development, Columbia University, College of Physicians & Surgeons, 701 West 168th Street, New York, 10032-2704, NY, USA

    Michael Lisby & Rodney Rothstein

  2. Center for Process Biotechnology, BioCentrum-DTU, Technical University of Denmark, Bldg. 223, Lyngby, DK-2800, Denmark

    Uffe H. Mortensen

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  1. Michael Lisby
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Correspondence to Rodney Rothstein.

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The authors declare no competing financial interests.

Supplementary information

Figure S1 Assembly and disassembly of spontaneous Rad52 foci.

Figure S2 Checkpoint arrest defect of mec1 sml1 cells. (PDF 2008 kb)

Figure S3 Induction of Rad52 foci by methyl methanesulfonate.

Figure S4 Insertion of an HO cut-site at iYCL016W and iYDR009C.

Figure S5 Insertion of an I-SceI cut-site at iYEL023C.

Figure S6 Insertion of an I-SceI cut-site at iYER186C.

Figure S7 Allele-replacement of GFP-LacI for YFP-LacI.

CONSTRUCTION OF YEAST STRAINS AND PLASMIDS

Table S1. Yeast strains

Table S2. Primers (DOC 36 kb)

Movie 1 (MOV 2339 kb)

Movie 2 (MOV 2894 kb)

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Lisby, M., Mortensen, U. & Rothstein, R. Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre. Nat Cell Biol 5, 572–577 (2003). https://doi.org/10.1038/ncb997

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