Rad51-mediated homologous recombination

(supported by grant Ha1374/4-3 from the Deutsche Forschungsgemeinschaft)

DNA double-strand breaks (DSBs), which may occur spontaneously or arise as a direct result of ionizing radiation or from exposure to DNA-damaging agents, are potentially lethal for the cell. Recombinational repair of DSBs is an essential process for genome integrity and propagation, that has been evolutionarily conserved from bacteria to man. In Escherichia coli, the vital steps of DNA recombination, homologous pairing and DNA strand exchange, are catalyzed by the RecA protein. Eukaryotic cells repair DSBs either by direct non-homologous end-joining of the broken ends or by homologous recombination. Homologous recombination also plays a fundamental role in the generation of genetic diversity during meiosis. Similar to RecA, both yeast and mammalian Rad51 proteins form nucleoprotein filaments on single-stranded (ss) DNA, mediating homologous pairing and strand-exchange reactions between ssDNA and homologous double-stranded DNA. In mammalian cells, non-homologous end-joining may be the primary pathway of DSB repair in G1 and early S phases, while homologous recombination may play a more dominant role in late S and G2 phases.

In cultured normal human cells, the HsRad51 protein is detected with immuno-fluorescent antibodies in multiple discrete foci in the nucleoplasm of a low number of cells. After DNA damage, the percentage of cells with focally concentrated Rad51 protein increases in a time- and dose-dependent manner. Rad51-foci positive cells are arrested during the cell cycle and undergo unscheduled DNA-repair synthesis (Haaf et al. 1995, 1999). Nuclear foci are formed at sites of DNA-damage induced ssDNA (Raderschall et al. 1999) and contain the ssDNA-binding replication protein A (Golub et al. 1998), which facilitates homologous pairing and DNA-strand exchange. The ssDNA-Rad51-RPA foci represent a repairosome-type assembly for recombinational DNA repair and identify a subset of cells that have entered the homologous recombination pathway. The abundance of Rad51 foci after induction of DNA damage by various agents is consistent with the view that homologous recombination plays an essential role in DSB repair in mammalian cells.

In contrast to E. coli RecA and yeast ScRad51, mammalian Rad51 appears to be necessary for cell proliferation and survival. Both the classical recombination/repair function and the newly acquired function(s) of Rad51 are studied in HsRad51-overexpressing cell lines and after antisense-inactivation of endogenous Rad51. Experimental evidence suggests that mammalian Rad51 protein participates in the regulation of a cell cycle checkpoint that allows the cell to repair damaged DNA and, thus, contributes to resistance to DNA damage-induced apoptosis. In this light, we study the interaction of Rad51 with the mitotic inhibitor p21 and other known cell cycle and apoptosis regulators. Expression profiling with cDNA microarrays and Western blotting experiments suggest that Rad51 may also be involved in the multi-step process by which tumor cells acquire immortality and metastatic growth. Rad51 over-expression may account, at least partially, for the resistance of many tumors to chemotherapy and thus may be a target for anti-sense-based gene therapy.