Fanconi anaemia (FA) is a hereditary, heterogeneous disease that is characterized by chromosomal instability, hypersensitivity to DNA cross-linking agents and cancer. Fifteen FA genes are identified, mutations in any of which are known to cause FA: FANCA, B, C, DI, D2, E, F, G, I, J, L, M, N, 0, and P. Cell lines defective for any FA gene show cellular and cytogenetic hypersensitivity to DNA inter-strand cross-linking agents such as mitomycin C (MMC). The FA proteins function through several complexes in a poorly defined, intricate phosphorylation-ubiquitination DNA repair network (the FA-BRCA pathway) that mediates the removal of inter-strand cross-links (ICL). ICL removal requires the action several different repair activities including cross-link unhooking, translesion synthesis and subsequent repair of the double-strand break (DSB) formed by homologous recombination. Critical for ICL repair is the FA core complex (comprising A, B, C, E, F, G, L and M proteins) that is essential for the mono- ubiquitination of FANCD2 and FANCI and formation of the ID complex. FANCG, one component of the FA core complex, is involved in at least two further distinct protein complexes that likely have independent functional roles to the core complex. The G-ERCC-XPF complex (FANCG-XPF-ERCCI) is required for inter-strand cross link (ICL) unhooking. The G-BRCA2 complex (FANCDIIBRCA2-FANCD2-FANCG-XRCC3) is hypothesised to be involved in modulating some aspect of homologous recombination repair (HRR). The aim of this study is to characterise the roles ofFANCG and the G-BRCA2 complex in ICL and DSB repair. MMC and phleomycin, that induces DNA strand breaks, were utilized to evaluate the efficiency of the HRR pathway in Chinese hamster ovary FANCG mutant cells (NM3) and human FANCA (GM6914) and F ANCD2 (PD20) fibroblasts. Drug hypersensitivity, chromosomal instability and HRR were evaluated using growth inhibition and clonal survival assays, metaphase analysis and a plasmid based HRR reporter assay respectively. Cell lines unable to form the G-BRCA2 complex are hypersensitive to phleomycin (in addition to MMC) and exhibit elevated chromosomal aberrations and reduced levels of HRR. This implicates the G-BRCA2 complex in the repair of both indirectly and directly induced DSB. Cell lines able to form the G-BRCA2 complex (such as FANCA-deficient cells) exhibit little or no hypersensitivity to phleomycin. The study also provides evidence that FANCD2 that is not mono-ubiquitinated has biological activity, specifically in the G-BRCA2 protein complex. The study provides important new insights into the molecular defects associated with FA and the role of the FA proteins in ICL and DSB repair.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:569198 |
| Date | January 2011 |
| Creators | Aladwani, Abdulaziz Mohamed |
| Publisher | University of Liverpool |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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