Functional characterization of the rad51 genes in Zea mays and their roles in Mu transposition

Abstract

<p>In Saccharomyces cerevisiae, Rad51p plays a central role in homologous recombination and DNA repair. Double mutants of the two Zea mays L. rad51 homologs are viable, but male sterile and have ~22% of normal seed set. Light microscopic analyses of male meiosis in these plants reveal: many chromosomes are unpaired at diakinesis and over 33% of quartets carry cells that either lack a nucleolus or have two nucleoli, indicating that non-disjunction occurs at both meiotic divisions. FISH analysis shows that 70% pachytene cells have paired 5S rDNA loci. Thus, maize RAD51 is required for efficient chromosome pairing and proper chromosome segregation in meiosis. FISH data also indicate that RAD51 is not essential for chromosome pairing. Consistent with that, surviving female gametes produced by double mutants are euploid and exhibit near-normal rates of meiotic crossovers. These results differ from those of Arabidopsis in which a rad51 mutant exhibits completely disrupted chromosome pairing during meiosis. Although maize rad51 double mutants develop well under normal condition, RAD51 function is critical for the repair of radiation-induced double-stranded breaks (DSBs) during early vegetative development;In late somatic cells rates of Mu insertion and excision are both high. In contrast, although high rates of insertion are observed in germinal cells, germinal excisions are recovered rarely. In RAD51- plants (i.e., rad51a, rad51b double mutants) rates of germinal derivatives from al-m5216 are 40-fold higher than controls. Most of the germinal derivatives involve deletions of the MuDR insertion and the al gene. This suggests that in wild-type germinal cells MuDR excisions are efficiently repaired via RAD51-directed homologous recombination with the sister chromatid, which replaces the excised MuDR. Two experiments suggest that RAD51 is also required for repairing Mu-induced DSBs during early vegetative development. First, a high proportion of Mu-active RAD51- mutants exhibit severe developmental defects. Second, ear sectors of germinal derivatives were recovered at a higher rate from RAD51- mutants than from controls. The higher rate at which partial deletions of the a1 locus were recovered indicates that the rad51 double mutant stock offers an attractive means to generate knock-out alleles for functional genomic studies.</p>