Date Published: January 20, 2004
Publisher: Public Library of Science
Author(s): Xuan Wang, James E Haber
Abstract: The single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) is essential for both DNA replication and recombination. Chromatin immunoprecipitation techniques were used to visualize the kinetics and extent of RPA binding following induction of a double-strand break (DSB) and during its repair by homologous recombination in yeast. RPA assembles at the HO endonuclease-cut MAT locus simultaneously with the appearance of the DSB, and binding spreads away from the DSB as 5′ to 3′ exonuclease activity creates more ssDNA. RPA binding precedes binding of the Rad51 recombination protein. The extent of RPA binding is greater when Rad51 is absent, supporting the idea that Rad51 displaces RPA from ssDNA. RPA plays an important role during RAD51-mediated strand invasion of the MAT ssDNA into the donor sequence HML. The replication-proficient but recombination-defective rfa1-t11 (K45E) mutation in the large subunit of RPA is normal in facilitating Rad51 filament formation on ssDNA, but is unable to achieve synapsis between MAT and HML. Thus, RPA appears to play a role in strand invasion as well as in facilitating Rad51 binding to ssDNA, possibly by stabilizing the displaced ssDNA.
Partial Text: Repair of double-strand breaks (DSBs) by homologous recombination involves the search for homology to locate an intact donor sequence. The search is successful when the broken DNA molecule basepairs with the homologous template, termed synapsis, and forms strand invasion intermediates of recombination. In budding yeast and other higher eukaryotes, this process requires both the Rad51 strand exchange protein and the single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) (Alani et al. 1992; Shinohara et al. 1992; Ogawa et al. 1993; Sung 1994; Symington 2002). RPA was first discovered through its essential role in SV40 DNA replication in vitro as a ssDNA-binding protein (Wold et al. 1989). The RPA complex forms a heterotrimer, which consists of three subunits of 70, 34, and 14 kDa, encoded by the RFA1, RFA2, and RFA3 genes, respectively (Wold 1997). Deletion of any of these genes leads to lethality in yeast (Heyer et al. 1990; Brill and Stillman 1991). The biological function of RPA was further demonstrated to be important in homologous recombination in Saccharomyces cerevisiae (Alani et al. 1992; Firmenich et al. 1995; Umezu et al. 1998) and in other aspects of DNA metabolism. Cells carrying a point mutation (K45E) in the largest subunit of RPA (rfa1-t11) are proficient for DNA replication, but their ability to perform mating-type (MAT) gene switching, single-strand annealing, and meiotic recombination is severely impaired (Umezu et al. 1998; Soustelle et al. 2002).
ChIP analysis provides a powerful tool for studying in vivo protein–DNA and protein–protein interactions. Using ChIP and related assays, we have demonstrated important roles of RPA during homologous recombination in vivo that could not have been known with certainty from in vitro studies. RPA is recruited to the DSB ends as soon as the DSB is detected on a Southern blot, and its binding precedes that of Rad51 (see Figures 2 and 4), which supports the idea that RPA is required to remove inhibitory secondary structures on ssDNA for Rad51 to polymerize across these regions (Sugiyama et al. 1997, 1998). This observation is also consistent with in vivo immunofluorescent staining results, suggesting that RPA foci appear earlier than Rad51 foci after irradiation (Golub et al. 1998; Gasior et al. 2001). Rad51 apparently displaces RPA from ssDNA, with the help of Rad52 (see Figure 3) and perhaps the Rad55/Rad57 auxiliary proteins. We note that our results are different from those reported by Wolner et al. (2003), who observed initial binding of RPA only after 45 min, whereas Rad51 was detected 25 min earlier, although it is not clear whether there is a statistically significant increase in Rad51 binding at the earliest time. In that assay, RFA1 was tagged with 13 Myc epitope tags, which may have altered its behavior. We believe our results are consistent with the fact that RPA has a higher-affinity constant for ssDNA and is present in much greater abundance in the cell (Heyer and Kolodner 1989; Mazin et al. 2000; Sugawara et al. 2003).