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The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy. ColloquiumAssembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosisStephen L.Gasior*†, Heidi Olivares*, Uy Ear*, Danielle M.Hari*, Ralph Weichselbaum*, and Douglas K.Bishop*†‡ Departments of *Radiation and Cellular Oncology, and †Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637 Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55 57 are required, whereas either Rad52 or Rad55/57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30°C but not 20°C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G1, consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication. Homologous recombination reactions promote repair of DNA ends formed by double-strand breaks (DSBs) and by replication fork collapse. Recombinational repair also allows cells to replicate past DNA lesions that block the progress of DNA polymerase. In phage T4, recombination is critical for initiating replication. In eukaryotes, homologous recombination is critical for accurate reductional segregation of chromosomes during meiosis. Finally, in prokaryotes, recombination allows horizontal transfer of alleles among and between bacteria and phage. At the center of homologous recombination are the recombinases, proteins that promote the formation of heteroduplex DNA. Of particular importance are the recombinases of the RecA family, including RecA in eubacteria, RadA in archea, Rad51 and Dmc1 in eukaryea, and the bacteriophage T4 UvsX protein. RecA-Like Recombinases Assemble on Single-Strand DNA (ssDNA). Several of the RecA recombinases have been shown to act by assembling into filaments on ssDNA (1–5). ssDNA tracts are formed by nucleolytic processing of DNA ends and by stalling of polymerase during DNA replication (6, 7). The nucleoprotein filaments formed by assembly of recombinases on ssDNA are capable of “searching” intact DNA duplexes for homologous regions (8–11). Location of a homologous duplex by the recombinase filament results in formation of a homologous joint between the ssDNA and the duplex and leads to strand exchange. During strand exchange, the ssDNA contained within the nucleoprotein filament forms Watson-Crick base pairs with the complementary strand of the “target” duplex, displacing the noncomplementary strand in the duplex. The hybrid DNA formed by strand exchange is further processed by repair polymerase and other recombination factors, eventually yielding two intact DNA duplexes (7, 8, 11, 12). Accessory Factors Act to Promote Assembly of Recombinase. Recombinases are able to promote strand exchange to form hybrid DNA in vitro without additional proteins. However, accessory factors can stimulate strand exchange. These factors can be divided into two broad classes: those that act before homology search by promoting assembly of recombinase filaments, and those that act during homology search and strand exchange. Assembly factors can, in turn, be divided into two classes: ssDNA-binding protein (ssb) and assembly “mediators.” Here we focus on the roles of assembly factors. A general model for the mechanism of recombinase assembly is shown in Fig. 1. ssbs are involved in multiple pathways of DNA metabolism, including replication, recombination, and repair, ssbs are abundant; the abundance of bacteriophage T4 gp32 protein is 1–3× 104 copies/infected cell (13), that of tetrameric Escherichia coli SSB about 1–2×103/cell (14), and that of the heterotrimeric RPA protein from eukaryotes about 104–105/cell (15). As their name implies, ssbs bind ssDNA specifically. The relative binding affinity (ssDNA/double-strand DNA) is at least 106 for gp32 (16), at least 108 for SSB (D.T.Lohman, personal communication), and 70-fold for RPA (17). ssbs can bind ssDNA in a cooperative manner, forming filaments that can readily saturate long stretches of ssDNA (18). ssbs can stimulate assembly of recombinases on ssDNA, but their ability to do so depends highly on reaction conditions (19–22). If an amount of ssb sufficient to saturate binding sites on ssDNA is added to strand exchange reactions before addition of recombinase, the reaction is inhibited. On the other hand, if recombinase is added first to such reactions, strand exchange activity is often stimulated compared with reactions containing no ssb. Inhibition of recombinase by ssb results from its ability to block initial binding of recombinase to ssDNA; ssb can outcompete recombinases as a consequence of their higher affinity and faster binding kinetics. A model that accounts for these observations is as follows, ssb competes with recombinase for initial binding sites on ssDNA. However, when recombinase This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA. Abbreviations: ssDNA, single-strand DNA; ssb, ssDNA-binding protein (generic); RPA, replication protein A; DSB, DNA double-strand break; CHO, Chinese hamster ovary; WT, wild type.
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