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, tRNA Ser UGAM, 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-tRNA Ser UGAM transcripts with elongated 3-oligo(U) tracts that are better substrates for La. A substantial fraction of pre-tRNA Ser UGAM 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.The 3Ј ends of tRNA and other RNA polymerase III (Pol III)-dependent genes contain dT n termination signals at which Pol III pauses and releases its RNA (6). The role of dT n extends beyond termination, since it provides a means to link Pol III transcripts to La, an abundant and ubiquitous nuclear phosphoprotein that binds these RNAs in a 3Ј-oligo(U) lengthdependent manner and promotes their posttranscriptional processing (27,32,37). Although 3Ј-U length heterogeneity has been well documented for Pol III transcripts (27), relatively little is known about the mechanisms involved and its functional significance. A model system that can be used to alter 3Ј-U length and study its consequences should be helpful in understanding functional connectivity between Pol III termination and posttranscriptional processing.La protein protects pre-tRNAs from 3Ј exonucleases and imposes order on posttranscriptional processing so that 5Ј processing precedes 3Ј processing for many pre-tRNAs (38). Finding the reverse order suggests that different pre-tRNAs use the La-independent and -dependent pathways to various degrees (25). While La-homologous protein (Lhp1p) is nonessential in the yeast Saccharomyces cerevisiae (and in Schizosaccharomyces pombe), its deletion causes lethality or growth deficiency in combination with mutations that impair base pairing or modification of certain pre-tRNAs, indicating that defective pre-tRNAs can be salvaged by La (5,7,21,38). Decay of hypomodified pre-tRNA i Met occurs via 3Ј adenylation and exonucleolytic degradation of pre-tRNA i Met in a process called nuclear surveillance (22) but can be rescued by excess Lhp1p (1, 5).

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 DSBinduced 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.

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. H OMOLOGOUS recombination is required forthe faithful repair of DNA double-strand breaks (DSBs) that arise during normal cellular processes or from exposure of cells to DNA-damaging agents. Central to the process of homologous recombination is the Rad51 protein, which facilitates synapsis and strand invasion into homologous duplex DNA (San Filippo et al. 2008). Rad51 belongs to the RecA family of homologous pairing proteins (Aboussekhra et al. 1992;Basile et al. 1992;Shinohara et al. 1992). Yeast and humans have two RecA homologs: Rad51 and the meiosis-specific Dmc1 (Bishop et al. 1992;San Filippo et al. 2008). In addition, the Saccharomyces cerevisiae RAD55 and RAD57 genes encode proteins with sequence similarity to RecA and Rad51 and are considered to be Rad51 paralogs (Kans and Mortimer 1991;Lovett 1994). Mutation of RAD51, RAD55, or RAD57 confers sensitivity of ionizing radiation (IR) and defects in mitotic and meiotic recombination, indicating that their functions are not redundant (Symington 2002). rad51 mutants generally exhibit more severe defects than rad55 or rad57 mutants in DSB-induced recombination assays; however, rad55 and rad57 mutants are more defective than rad51 in some assays that measure spontaneous recombination between repeated sequences (Rattray and Symington 1995;Mozlin et al. 2008).The molecular details of homologous recombination are largely based on genetic, physical, and cytological studies of DSB repair (DSBR) and on biochemical characterization of purified proteins (Paques and Haber 1999;Sugawara et al. 2003;Lisby et al. 2004;San Filippo et al. 2008). The single-stranded DNA (ssDNA)-binding protein, replication protein A (RPA), initially binds ssDNA that forms by nucleolytic processing of DNA ends at DSBs. In vitro, RPA has been shown to be inhibitory to Rad51 binding to ssDNA, but this inhibition c...