Baumann, Peter; (1998) Biochemical characterisation of the human RAD51 recombinase. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In Saccharomyces cerevisiae mutations in the RAD51 gene result in sensitivity to ionising radiation and defects in mitotic and meiotic recombination. Homologues of RAD51 have been identified in a wide range of higher eukaryotes including humans. Purification and biochemical characterisation of yeast and human Rad51 revealed extensive structural and functional similarities with the Escherichia coli RecA protein, a key component of the bacterial recombination machinery. In the work described here, human Rad51 (hRad51) was expressed in E. coli and baculovirus infected insect cells and was purified by a method involving spermidine-mediated precipitation and conventional column chromatography. Human Rad51 was found to form nucleoprotein filaments on single- and double-stranded DNA and to display DNA-dependent ATPase activity. Consistent with a role in recombination and DNA repair, hRad51 promoted homologous pairing and strand transfer reactions between circular single-stranded and linear duplex DNA. Joint molecule formation was dependent on ATP and occurred with 3' to 5' polarity with respect to the single stranded DNA on which the Rad51 filament was originally formed. The extent of strand transfer observed in the presence of hRad51 was limited to - 1.2 kb, indicating that additional factors are required for extensive heteroduplex formation in higher organisms. The human single-stranded DNA binding protein, hRP-A, has multiple functions in replication, recombination and DNA repair. Using in vitro recombination assays, hRP-A was found to stimulate hRad51-mediated pairing reactions. The effect of hRP-A is consistent with a role in the removal of DNA secondary structures from the single-stranded DNA, thereby facilitating the formation of continuous hRad51 filaments. Studies in S. cerevisiae indicated physical and functional interactions between the Rad51 and Rad52 proteins. Here it is shown that human Rad52 stimulates hRad51-mediated homologous pairing, most likely by affecting Rad51 filament formation.

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