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, 26 (2), 678-88

Rec2 Interplay With Both Brh2 and Rad51 Balances Recombinational Repair in Ustilago Maydis

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Rec2 Interplay With Both Brh2 and Rad51 Balances Recombinational Repair in Ustilago Maydis

Milorad Kojic et al. Mol Cell Biol.

Abstract

Rec2 is the single Rad51 paralog in Ustilago maydis. Here, we find that Rec2 is required for radiation-induced Rad51 nuclear focus formation but that Rec2 foci form independently of Rad51 and Brh2. Brh2 foci also form in the absence of Rad51 and Rec2. By coprecipitation from cleared extracts prepared from Escherichia coli cells expressing the proteins, we found that Rec2 interacts physically not only with Rad51 and itself but also with Brh2. Transgenic expression of Brh2 in rec2 mutants can effectively restore radiation resistance, but the frequencies of spontaneous Rad51 focus formation and allelic recombination are elevated. The Dss1-independent Brh2-RPA70 fusion protein is also active in restoring radiation sensitivity of rec2 but is hyperactive to an extreme degree in allelic recombination and in suppressing the meiotic block of rec2. However, the high frequency of chromosome missegregation in meiotic products is an indicator of a corrupted process. The results demonstrate that the importance of Rec2 function is not only in stimulating recombination activity but also in ensuring that recombination is properly controlled.

Figures

FIG. 1.
FIG. 1.
Focus formation. Wild-type (WT) (UCM350) cells expressing GFP-Rad51, Rec2, or Brh2 were viewed by differential interference contrast (DIC) imaging and fluorescence microscopy without fixation. (A) Cells were examined for focus formation 4 h after irradiation with UV (30 J/m2). Cells with representative Brh2, Rad51, or Rec2 foci are shown. Typically, cells had one to two foci. Bar indicates 3 μm. (B) The fraction of cells with foci at each time point was determined after counting approximately 200 cells. The cumulative representations of each distribution are shown on the right. (C) rec2 (UCM54), brh2 (UCM565), or rad51 (UCM628) mutant cells expressing GFP-Rad51, Rec2, or Brh2 were examined for foci at 2 and 4 h as described above. +, competent in focus formation; −, no focus formation. (D) Survival of rad51, rec2, and brh2 mutant strains expressing GFP-tagged or untagged Rad51, Rec2, or Brh2, respectively, after irradiation with UV (120 J/m2) and, in the case of rad51, with gamma rays (400 Gy). Serial 10-fold dilutions of cell suspensions were spotted from left to right as shown.
FIG. 2.
FIG. 2.
Rec2 interactions. E. coli strains expressing the indicated proteins were processed as described in Materials and Methods. After specific elution from affinity resins, samples were electrophoresed in 10% polyacrylamide gels containing sodium dodecyl sulfate and analyzed by Western blotting. (A) His-Rec2 and His-Rec2 NT (Rec21-174) interact with Rad51. NTA, nitrilotriacetic acid. (B) MBP-Brh2 interacts with His-Rec2 and His-Rec2 NT (Rec21-174). (C) His-Rad51 interacts with MBP-BRC (Brh2260-330) but not with MBP-BRCpm (point mutations FT294 and 296AA). (D) His-Rec2 interacts with MBP-BRC (Brh2260-330) but not with MBP-BRCpm (point mutations FT294 and 296AA). (E) His-Rec2 interacts with MBP-Rec2.
FIG. 3.
FIG. 3.
Suppression of rec2 radiation sensitivity and restoration of Rad51 focus formation. (A) rec2 strains (UCM54) expressing Rad51, Brh2, Brh2-RPA70, RPA70, or Rec2 were tested for survival after irradiation with UV (120 J/m2) or gamma rays (400 Gy). Serial 10-fold dilutions of cell suspensions were spotted from left to right as shown. wt, wild type. (B) rad51 (UCM628) or brh2 (UCM565) strains expressing untagged Rad51, Rec2, or Brh2 were tested for survival after irradiation with UV as described above. (C) GFP-Rad51 was expressed in rec2 strains with integrated transgenes expressing Brh2, Brh2-RPA70, or Rec2. Cells were monitored for Rad51 focus formation 60 min after administering a 40-Gy dose of gamma rays.
FIG. 4.
FIG. 4.
Hyperactive allelic recombination. rec2/rec2 homozygous diploid strains (UCM110) heteroallelic at the ino1 locus and expressing Rec2, Brh2, or Brh2-RPA70 from a transgene, plus a wild-type control strain (UCM96), were irradiated with increasing doses of UV light and plated onto minimal medium to score Ino+ recombinants or onto YEPS to monitor cell viability. Five independent cultures of each strain were tested. The mean frequencies and standard deviations are shown. wt, wild type.
FIG. 5.
FIG. 5.
Meiotic rescue. Teliospores obtained from wild-type and rec2/rec2 homozygous crosses with or without an integrated transgene expressing Brh2-RPA70 were germinated on YEPS at 30°C for 38 h. (A) Teliospores (wild type) before germination viewed at ×1,000 magnification. (B) rec2/rec2 teliospores at 38 h after germination, arrested after promycelium formation. (C) rec2/rec2/Brh2-RPA70 microcolonies at 38 h. (D) Wild-type microcolonies at 38 h. Microcolonies from rec2/rec2/Brh2-RPA70 (E) and the wild type (F) were collected and dispersed to single cells which were spread onto medium containing charcoal to enable fuzz formation. Colonies appearing after incubation for 3 days were recorded. A section from petri dishes with Fuz+ (white) and Fuz (gray) colonies is shown.

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