Homologous recombination in planta is stimulated in the absence of Rad50

Abstract

Chromosomal double-strand DNA breaks must be repaired; in the absence of repair the resulting acentromeric (and telomereless) fragments may be lost and/or the broken DNA ends may recombine causing general chromosomal instability. The Rad50/Mre11/Xrs2 protein complex acts at DNA ends and is implicated in both homologous and non-homologous recombination. We have isolated a rad50 mutant of the plant Arabidopsis thaliana and show here that it has a somatic hyper-recombination phenotype in planta. This finding supports the hypothesis of a competition between homologous and illegitimate recombination in higher eukaryotes. To our knowledge, this is the first direct in vivo support for the role of this complex in chromosomal recombination in a multicellular organism and the first description of a mutation of a known gene leading to hyper-recombination in plants.

Arabidopsis RAD51C Gene Is Important for Homologous Recombination in Meiosis and Mitosis

“…Rad51C deficiency also results in reduced frequencies of somatic recombination in Rad51C knockout DT40 and CL-V4B cells Drexler et al, 2004). To determine the direct involvement of AtRad51C in HR, we measured the frequencies of somatic recombination in the atrad51C mutant with GUS recombination reporter system developed by the group of B. Hohn (Swoboda et al, 1994;Gherbi et al, 2001). Using this system, we confirmed that AtRad51C required the efficient recombination in somatic cells (Table I; Fig.…”

“…We used both direct-and inverted-repeat recombination reporter lines (1406 and 1415) for the evaluation of somatic HRF in the atrad51C mutant. In the case of the direct repeat, the production of a functional recombined GUS gene occurs by gene conversion/crossover or single-strand annealing mechanisms; on the other hand, only a gene conversion/crossover produced a functional recombined GUS gene for the inverted repeat (Gherbi et al, 2001;Dubest et al, 2004). Somatic HRF in atrad51C reduced in both direct-and invertedrepeat reporter lines at similar rates under or without genotoxic stresses (Table I).…”

“…For further characterization of AtRad51 function in HR, the atrad51C plants were monitored for somatic HR using an in planta recombination assay with a reporter gene consisting of two overlapping fragments of the b-glucuronidase (GUS) gene separated by a hygromycin-resistant marker (Swoboda et al, 1994, Gherbi et al, 2001). The GUS sequences have an Figure 5.…”

“…Fifteen-day-old plants untreated and treated with 30 mM and 50 mM cisplatin were transferred on MS agar medium without cisplatin, and cultured for additional 6 to 7 d. Top of each section, wild-type plants; center of each section, atrad51C plants; bottom of each section, overlap of 618 bp, and recombination event between the two overlapping sequences can produce a functional GUS gene resulting in blue-colored GUS spots in cells, which are detected by histochemical staining with 5-bromo-4-chloro-3-indoryl glucuronide. We used the two GUS recombination reporter lines, 1406 (directrepeat recombination reporter line) and 1415 (invertedrepeat recombination reporter line), for in planta recombination assay (Gherbi et al, 2001). The GUS recombination reporter line 1406 or 1415 was crossed with the heterozygous AtRAD51C 1/2 plant.…”

Reevaluation of the Reliability and Usefulness of the Somatic Homologous Recombination Reporter Lines

“…The articles reporting the use of this line cite one or more of the following sources: Tinland et al (1994), Gherbi et al (2001), Fritsch et al (2004), or Lucht et al (2002, yet none of these cited articles offers any detailed information on this line. Therefore, the origin of this line is unclear, along with the details of transgene copy number and location.…”