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Title: The role of RAD51-like genes in the repair of DNA damage in mammalian cells
Author: French, Catherine A.
ISNI:       0000 0001 3484 1535
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2003
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Homologous recombination (HR) is essential for the repair of severe forms of DNA damage such as double strand breaks and interstrand cross-links. The highly conserved RAD51 protein is central to this process and forms nucleoprotein filaments on ssDNA that catalyse strand exchange. In mammalian cells, five genes that share sequence homology with RAD51 have been identified; XRCC2, XRCC3, RAD5JL1, RAD51L2 and RAD51L3. Interactions amongst the RAD51-like proteins have been demonstrated but at present little is known about their functions in HR. Progress has been hampered in the cases of RAD5lLl, -L2 and

-L3, which were identified through database homology searches, because of a lack of suitable mutant cell lines in which to study them.

Cultured mammalian cell lines, selected for sensitivity to DNA-damaging agents, have been used to clone and characterise the function of genes from several DNA repair pathways. The irs3 cell line was isolated from the Chinese hamster line V79-4 in a screen for radiosensitive mutants and has a unique genetic defect. irs3 cells are sensitive to X-rays (2-fold), ethyl methanesulphonate (2.5-fold) and especially mitomycin-C (MMC) (7-fold), as well as showing chromosomal instability. In this study, the sensitivity of irs3 to DNA-damaging agents was found to be substantially complemented by expression of the human RAD51L2 gene, but not other RAD51-like genes or RAD51 itself. An increased level of spontaneous chromosome aberrations was also complemented by RAD51L2 cDNA expression. irs3 cells were found to have little or no RAD51L2 cDNA and RAD51L2 protein. This is likely due to a G → T base substitution at the first nucleotide of exon 6 in the hamster RAD51L2 gene, which encodes an amino acid change of Val to Phe and disrupts the exon 6 3' acceptor splice site. In collaboration with Dr. Jean-Yves Masson and Dr. Steve West (Cancer Research UK, Clare Hall Laboratories, London), two RAD51 -like protein complexes were shown to exist in hamster cells as they do in human cells. Strikingly, neither complex is formed in irs3. Site-directed mutagenesis of the invariant lysine in the RAD51L2 Walker A box ATP-binding domain showed that this residue is required for MMC resistance. Consistent with a role in HR, -the RAD51L2 protein was found to localise to the nucleus. This localisation was dependent on the eleven amino acids at the C-terminus of the protein. irs3 cells showed a reduced number of MMC-induced sister chromatid exchanges compared to wild type cells. irs3 cells were also found to be defective in damage-dependent RAD51 focus formation, providing the first evidence that RAD51L2 is involved in repair via RAD51-dependent HR. Using the I-Scel endonuclease to generate a double strand break, it was found that gene conversion is decreased 7-fold in irs3 compared to wild type cells. In summary, this study provides data which identify irs3 as a RAD51L2-deficient cell line and demonstrate that RAD51L2 plays a key role in RAD51-dependent HR processes.

Supervisor: Thacker, John Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: DNA damage ; DNA-binding proteins