Physical Interaction between Recombinational Proteins Rhp51 and Rad22 in Schizosaccharomyces pombe

Abstract

In eukaryotes, Rad51 and Rad52 are two key components of homologous recombination and recombinational repair. These two proteins interact with each other. Here we investigated the role of interaction between Rhp51 and Rad22, the fission yeast homologs of Rad51 and Rad52, respectively, on the function of each protein. We identified a direct association between the two proteins and their self-interactions both in vivo and in vitro. We also determined the binding domains of each protein that mediate these interactions. To characterize the role of Rhp51-Rad22 interaction, we used random mutagenesis to identify the mutants Rhp51 and Rad22, which cannot interact each other. Interestingly, we found that mutant Rhp51 protein, which cannot interact with either Rad22 or Rti1 (G282D), lost its DNA repair ability. In contrast, mutant Rad22 proteins, which cannot specifically bind to Rhp51 (S379L and P381L), maintained their DNA repair ability. These results suggest that the interaction between Rhp51 and Rad22 is crucial for the recombinational repair function of Rhp51. However, the significance of this interaction on the function of Rad22 remains to be characterized further. Double strand breaks (DSBs)1 in chromosome are very harmful, and the failure in repair of DSB results in severe genomic instabilities that can lead to cell death or, in higher eukaryotes, to cancer (1). In eukaryotes, two major pathways have been known to deal with DSBs (2). The nonhomologous end-joining pathway joins adjacent broken DNA ends, resulting in errors in the junction region. In contrast, the homologous recombination (HR) pathway takes advantage of the undamaged homologous DNA strands, resulting in accurate repair of DSBs.In Saccharomyces cerevisiae, RAD52 epistasis group genes are involved in HR pathway (3). These genes, including RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RPA, MRE11, and XRS2, are well conserved throughout eukaryotes. In addition to these genes, there are several species-specific genes involved in HR, such as RAD59 (4) and RDH54/TID1 (5) in S. cerevisiae, rti1ϩ /rad22B ϩ (6) in Schizosaccharomyces pombe, and BRCA1 (7), BRCA2 (8), Rad51B-D, Xrcc2, and Xrcc3 (9) in mammals.RAD52 is the only gene among the RAD52 epistasis group that is required for virtually all homologous recombination events (3). Purified S. cerevisiae and human Rad52 proteins (ScRad52 and HsRad52, respectively) have an annealing activity of two complementary single-stranded DNAs (10), and they promote the strand exchange activity of Rad51 in the presence of RPA (11). In addition, HsRad52 and S. pombe Rad52 (Rad22) appear to bind to DSBs (12, 13). Rad51 is an eukaryotic structural and functional counterpart of Escherichia coli RecA (14). Purified ScRad51 and HsRad51 have the homologous pairing/ strand exchange activity that is the core catalytic activity of HR (15). Rti1/Rad22B (hereafter referred to simply as Rti1) is another Rad52 homolog found in S. pombe (6, 16). Deletion of both rad22 ϩ and rti1 ϩ leads to more severe defects compared with each single mu...

Reconstitution of DNA Strand Exchange Mediated by Rhp51 Recombinase and Two Mediators

“…However, since the RPA levels in the unbound fractions increased only slightly when Rad22 was also included (Figure 4H), the observed increase of Rhp51 in Figure 4I may be due to Rhp51 bound to RPA-coated css beads via Rad22. It has been reported that Rad22 interacts with both Rhp51 and RPA [31,32], and the ternary complex can also be detected in vitro by coimmunoprecipitation (unpublished data). However, RPA displacement mediated by Rhp51 with Swi5-Sfr1 was further stimulated by coincubation with Rad22 (Figure 4G).…”

The Rad52 Homologs Rad22 and Rti1 ofSchizosaccharomyces pombeAre Not Essential for Meiotic Interhomolog Recombination, but Are Required for Meiotic Intrachromosomal Recombination and Mating-Type-Related DNA Repair

“…The N-terminal ends of Rad52 homologs are required for self-interaction, DNA binding, and annealing and thus play a key role in the function of these proteins (Asleson et al 1999). The C-terminal regions are required for interaction with Rad51 and are more species specific (Milne and Weaver 1993;Kagawa et al 2002;W. J. Kim et al 2002) (Figure 1).…”

Mus81, Rhp51(Rad51), and Rqh1 Form an Epistatic Pathway Required for the S-Phase DNA Damage Checkpoint

“…Rad22 is the fission yeast homologue to budding yeast Rad52, which is responsible for loading Rhp51 onto 3Ј end of ssDNA (Kim et al, 2002). Some recombination events in fission yeast such as single-strand annealing are Rad22 de- pendent but Rhp51 independent .…”