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Brh2-Dss1 Interplay Enables Properly Controlled Recombination in Ustilago maydis

Milorad Kojic,^1, Qingwen Zhou,^1, Michael Lisby,^2,, and William K. Holloman^1*

Department of Microbiology and Immunology, Hearst Microbiology Research Center, Cornell University Weill Medical College,¹ Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, New York²

Received 19 November 2004/ Returned for modification 8 December 2004/ Accepted 27 December 2004

Brh2, the BRCA2 homolog in Ustilago maydis, functions in recombinational repair of DNA damage by regulating Rad51 and is, in turn, regulated by Dss1. Dss1 is not required for Brh2 stability in vivo, nor for Brh2 to associate with Rad51, but is required for formation of green fluorescent protein (GFP)-Rad51 foci following DNA damage by gamma radiation. To understand more about the interplay between Brh2 and Dss1, we isolated mutant variants of Brh2 able to bypass the requirement for Dss1. These variants were found to lack the entire C-terminal DNA-Dss1 binding domain but to maintain the N-terminal region harboring the Rad51-interacting BRC element. GFP-Rad51 focus formation was nearly normal in brh2 mutant cells expressing a representative Brh2 variant with the C-terminal domain deleted. These findings suggest that the N-terminal region of Brh2 has an innate ability to organize Rad51. Survival after DNA damage was almost fully restored by a chimeric form of Brh2 having a DNA-binding domain from RPA70 fused to the Brh2 N-terminal domain, but Rad51 focus formation and mitotic recombination were elevated above wild-type levels. The results provide evidence for a mechanism in which Dss1 activates a Brh2-Rad51 complex and balances a finely regulated recombinational repair system.

Supplemental material for this article may be found at http://mcb.asm.org/.

M.K., Q.Z., and M.L. contributed equally to this work.

Present address: Department of Genetics, Institute of Molecular Biology, University of Copenhagen, Copenhagen, Denmark.

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