ABSTRACT
The Rad51 paralogs Rad55 and Rad57 form a heterodimer required to mediate the formation and/or stabilization of the Rad51 filament. To further characterize the function of Rad55-Rad57, we used a combination of rad57 partial suppressors to determine whether the DNA repair and recombination defects of the rad57 mutant could be completely suppressed. The combination of all suppressors, elevated temperature, srs2, rad51-I345T, and mating-type (MAT) heterozygosity resulted in almost complete suppression of the rad57 mutant defect in the recruitment of Rad51 to DNA-damaged sites, as well as survival in response to ionizing radiation and camptothecin. In a physical assay to monitor the kinetics of double-strand-break (DSB)-induced gene conversion, the rad57 mutant defect was effectively suppressed by srs2 and MAT heterozygosity, but these same suppressors failed to suppress the spontaneous recombination defect. Thus the Rad55-Rad57 heterodimer appears to have a unique function in spontaneous recombination that is not essential for DSB repair. Furthermore, we investigated the currently unknown mechanism of rad57 suppression by MAT heterozygosity and found that it is independent of DNL4.
Subject(s)
Adenosine Triphosphatases/genetics , DNA Breaks, Double-Stranded , DNA Repair Enzymes/genetics , DNA Repair/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/physiology , DNA Helicases/genetics , DNA Repair Enzymes/metabolism , DNA Repair Enzymes/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Down-Regulation/genetics , Gene Conversion/genetics , Genes, Mating Type, Fungal/physiology , Heterozygote , Models, Biological , Mutant Proteins/genetics , Mutant Proteins/physiology , Organisms, Genetically Modified , Protein Multimerization , Recombination, Genetic/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/physiologyABSTRACT
Rad51 requires a number of other proteins, including the Rad51 paralogs, for efficient recombination in vivo. Current evidence suggests that the yeast Rad51 paralogs, Rad55 and Rad57, are important in formation or stabilization of the Rad51 nucleoprotein filament. To gain further insights into the function of the Rad51 paralogs, reporters were designed to measure spontaneous or double-strand break (DSB)-induced sister or nonsister recombination. Spontaneous sister chromatid recombination (SCR) was reduced 6000-fold in the rad57 mutant, significantly more than in the rad51 mutant. Although the DSB-induced recombination defect of rad57 was suppressed by overexpression of Rad51, elevated temperature, or expression of both mating-type alleles, the rad57 defect in spontaneous SCR was not strongly suppressed by these same factors. In addition, the UV sensitivity of the rad57 mutant was not strongly suppressed by MAT heterozygosity, even though Rad51 foci were restored under these conditions. This lack of suppression suggests that Rad55 and Rad57 have different roles in the recombinational repair of stalled replication forks compared with DSB repair. Furthermore, these data suggest that most spontaneous SCR initiates from single-stranded gaps formed at stalled replication forks rather than DSBs.
Subject(s)
DNA-Binding Proteins/metabolism , Rad51 Recombinase/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid , Sister Chromatid Exchange/genetics , Adenosine Triphosphatases , DNA Breaks, Double-Stranded/drug effects , DNA Breaks, Double-Stranded/radiation effects , DNA Breaks, Single-Stranded/drug effects , DNA Breaks, Single-Stranded/radiation effects , DNA Repair Enzymes , Diploidy , Gene Conversion/drug effects , Gene Conversion/radiation effects , Heterozygote , Mating Factor , Models, Genetic , Mutagens/pharmacology , Mutation/drug effects , Peptides/metabolism , Recombinant Fusion Proteins/metabolism , Repetitive Sequences, Nucleic Acid/genetics , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/radiation effects , Sister Chromatid Exchange/drug effects , Sister Chromatid Exchange/radiation effects , Suppression, Genetic/drug effects , Suppression, Genetic/radiation effects , Temperature , Ultraviolet RaysABSTRACT
Termination by RNA polymerase III (Pol III) produces RNAs whose 3' oligo(U) termini are bound by La protein, a chaperone that protects RNAs from 3' exonucleases and promotes their maturation. Multiple reports indicate that yeasts use La-dependent and -independent pathways for tRNA maturation, with defective pre-tRNAs being most sensitive to decay and most dependent on La for maturation and function. The Rpc11p subunit of Pol III shows homology with the zinc ribbon of TFIIS and is known to mediate RNA 3' cleavage and to be important for termination. We used a La-dependent opal suppressor, tRNASerUGAM, which suppresses ade6-704 and the accumulation of red pigment, to screen Schizosaccaromyces pombe for rpc11 mutants that increase tRNA-mediated suppression. Analyses of two zinc ribbon mutants indicate that they are deficient in Pol III RNA 3' cleavage activity and produce pre-tRNASerUGAM transcripts with elongated 3'-oligo(U) tracts that are better substrates for La. A substantial fraction of pre-tRNASerUGAM contains too few 3' Us for efficient La binding and appears to decay in wild-type cells but has elongated oligo(U) tracts and matures along the La-dependent pathway in the mutants. The data indicate that Rpc11p limits RNA 3'-U length and that this significantly restricts pre-tRNAs to a La-independent pathway of maturation in fission yeast.