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Title: Effects of overexpressing wild-type and variant Rad52 on homologous recombination in human cells
Author: Hong, Julia Mai-Ling
ISNI:       0000 0004 2711 7073
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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Mitotic homologous recombination (HR) stabilises the genome by repairing harmful double stranded DNA (dsDNA) breaks. Rad52 promotes the annealing of complementary DNA strands and binds to Rad51 suggesting that Rad52 acts in both strand invasion (SI) and single strand annealing (SSA) HR pathways. Overexpression of Rad52 in mammalian cells can greatly stimulate HR, but the degree of stimulation varies widely between studies and inhibitory effects have also been described. The in vivo roles for Rad52 in mammalian cells are thus poorly understood as is the potential of using Rad52 as a tool to promote genome engineering methods [i.e. gene targeting (GT)]. Here I have systematically compared the effects of overexpressing wild-type (wt) and mutant Rad52 proteins on cell viability and various HR assays in human cells. Mutants were designed to test the potential involvement of defined domains/residues in any such effects. Human Rad52 (hRad52) and its derivatives negatively affected cell viability and proliferation which correlated with the presence of C-terminal residues 331-418, rather than, as expected, the Rad51 binding domain which limited inhibitory effects. Negative effects with yeast Rad52 (scRad52) were not observed. Consistent with previous findings, hRad52 inhibited GT, however, this was converted to a stimulatory effect when residues 331-418 were removed. When single stranded oligonucleotide (ssO) templates were used for GT, both hRad52 and its derivatives were stimulatory. These results are consistent with SI and SSA models for dsDNA- and ssO-mediated GT, respectively, and suggest that residues 331-418 cause a dominant-negative effect on SI and their removal promotes the strand annealing activity of the N-terminal domain. ScRad52 stimulated and inhibited GT with ds and ss templates, respectively. Altogether, these results provide the first evidence that truncated forms of hRad52 may serve as useful tools for promoting GT using both ds and ss DNA templates in human cells.
Supervisor: Porter, Andy Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral