Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death

Abstract

Yeast rad51 mutants are viable, but extremely sensitive to γ-rays due to defective repair of double-strand breaks. In contrast, disruption of the murine RAD51 homologue is lethal, indicating an essential role of Rad51 in vertebrate cells. We generated clones of the chicken B lymphocyte line DT40 carrying a human RAD51 transgene under the control of a repressible promoter and subsequently disrupted the endogenous RAD51 loci. Upon inhibition of the RAD51 transgene, Rad51 -cells accumulated in the G 2 /M phase of the cell cycle before dying. Chromosome analysis revealed that most metaphase-arrested Rad51 -cells carried isochromatid-type breaks. In conclusion, Rad51 fulfils an essential role in the repair of spontaneously occurring chromosome breaks in proliferating cells of higher eukaryotes.

Chk2 is required for optimal mitotic delay in response to irradiation-induced DNA damage incurred in G2 phase

“…The sequence of the PCR primers is available on request. Two separate targeting vectors containing a common 1.0 kb left and 3.5 kb right arm of homology were synthesized by long-range PCR and cloned into Bluescript (Stratagene) flanking either neomycin or puromycin drug selection cassettes (Sonoda et al, 1998). The targeting constructs were designed to delete both the Chk2 FHA and kinase domains so that any residual truncated polypeptide encoded by the targeted alleles would be non-functional.…”

“…Asynchronous cultures of WT, Chk2À/À and Chk2Rev2 cells were treated with colcemid for 3 h and metaphase spreads prepared and scored by light microscopy for isochromatid breaks and gaps affecting macrochromosomes as described previously (Sonoda et al, 1998). An example of a Chk2À/À metaphase with an isochromatid break is shown in Figure 7d.…”

Gene targeting is enhanced in human cells overexpressing hRAD51

“…27,28 Equivalent experiments with RAD51 have not been possible because of the lethal effects of inactivation. 29 Resistance to DNA damage, presumably reflecting DNA repair by homologous recombination, is also severely reduced upon RAD54 or RAD51 inactivation. 25,26,30 Overexpression of S. cerevisiae RAD52 in human cells, 31 of human RAD52 in monkey cells 32 and of hamster RAD51 in CHO cells 33 has been shown to promote ionising radiation resistance and intrachromosomal or extrachromosomal homologous recombination.…”

XRCC4 in G1 suppresses homologous recombination in S/G2, in G1 checkpoint-defective cells

“…The second and alternative strategy is non-homologous end joining (NHEJ), which ligates the DNA ends without requiring sequence homologies between the two interacting molecules. Defects in HR or in NHEJ can lead to genome instability and tumorigenesis (Liu et al, 1998;Sonoda et al, 1998;Difilippantonio et al, 2000;Ferguson et al, 2000a;Bertrand et al, 2003). However, both functional HR and NHEJ can be responsible for genome rearrangements: (i) functional HR between repeated sequences dispersed through the genome can also lead to genome rearrangements (Purandare and Patel, 1997;Richardson and Jasin, 2000b;Bertrand et al, 2004), and (ii) functional Ku autoantigen protein (KU)-dependent as well as KU-independent NHEJ can generate genetic rearrangements (Guirouilh-Barbat et al, 2004).…”