Homologous recombination deficiency leads to profound genetic instability in cells derived from Xrcc2-knockout mice.

DNA damage such as double-strand breaks presents severe difficulties for the cell to repair, especially if genetic stability is to be preserved. Recombination of the damaged DNA molecule with an undamaged homologous sequence provides a potential mechanism for the high-fidelity repair of such damage, and genes encoding homologous recombination (HR) proteins have been identified in mammalian cells. Xrcc2 is a protein with homology to Rad51, the core component of HR, but with a nonredundant role in damage repair. Here, we make the first study of the consequences of knocking out one or both copies of the Xrcc2 gene in mouse cells. In addition to growth arrest and sensitivity to agents causing severe DNA damage, we show that order-of-magnitude higher levels of chromosomal alterations are sustained in primary or immortal Xrcc2(-/-) embryonic fibroblasts. Using spectral karyotyping, we find that aneuploidy and complex chromosome exchanges, including an unexpectedly high frequency of homologue exchanges, are hallmarks of Xrcc2 deficiency. In addition, we find evidence for mild haploinsufficiency of Xrcc2. These responses are linked to several indicators of reduced HR in Xrcc2(-/-) cells, including a 30-fold reduction in gene conversion and reduced levels of Rad51-focus formation and of sister-chromatid exchange. Our data have similarities to recent studies of the disruption of breast cancer-predisposing (Brca) genes in mouse cells and are contrasted to analyses of cells carrying disruptions of genes in the other main pathway for double-strand break repair, nonhomologous end joining.

Transmissible and nontransmissible complex chromosome aberrations characterized by three-color and mFISH define a biomarker of exposure to high-LET alpha particles.

Insertions have been proposed as potential stable biomarkers of chronic high-LET radiation exposure. To examine this in vitro, we irradiated human peripheral blood lymphocytes in G(0) with either 50 cGy (238)Pu alpha particles (LET 121.4 keV/microm) or 3 Gy 250 kV X rays and stimulated their long-term culture up to approximately 22 population doublings postirradiation. Mitotic cells were harvested at regular intervals throughout this culture period and were assayed for chromosome aberrations using the techniques of three-color and 24-color mFISH. We observed the stable persistence of transmissible-type complex rearrangements, all involving at least one insertion. This supports the hypothesis that insertions are relevant indicators of exposure to high-LET radiation. However, one practical caveat of insertions being effective biomarkers is that their frequency is low due to the complexity and cell lethality of the majority of alpha-particle-induced complexes. Therefore, we propose a "profile of damage" that relies on the presence of insertions, a low frequency of stable simple reciprocal translocations (2B), and, significantly, the complexity of the damage initially induced. We suggest that the complexity of first- and second-division alpha-particle-induced nontransmissible complex aberrations reflects the structure of the alpha-particle track and as a consequence adds radiation-quality specificity to the biomarker, increasing the signal:noise ratio of the characteristic 2B:insertion ratio.

Clustered DNA damage leads to complex genetic changes in irradiated human cells.

Densely ionizing radiations interact with DNA to cause heavily clustered sites of damage that are difficult to repair correctly. We have been able to determine for the first time the breakpoints of several very large deletions induced by densely ionizing radiation in diploid human cells and show that damage clustering is reflected in the complexity of mutations. Intra- and interchromosomal insertions and inversions occur at the sites of some large deletions. Short sequence repeats are commonly found at the breakpoints, showing that microhomologies help patch damage sites. We suggest that novel fragments found in complex rearrangements derive from other sites of radiation damage in the same cell. These transmissible molecular changes are echoed by visible chromosome rearrangements many days after irradiation and are likely to contribute significantly to the carcinogenic properties of densely ionizing radiations.

Aneuploidy, centrosome activity and chromosome instability in cells deficient in homologous recombination repair.

Chromosome instability and loss or gain of chromosomes are changes characteristic of many tumour cells and human disorders. However, the mechanism of these changes has not yet been fully determined. We have recently shown that hamster cell lines deficient in homologous recombination repair (HRR) genes XRCC2 and XRCC3 have an elevated frequency of aneuploidy compared with wild-type cells and mutant cells transfected with the appropriate human gene. In addition, XRCC2 and XRCC3 deficient hamster cell lines show a high frequency of multiple centrosomes and abnormal spindle formation. Cells deficient in HRR show a high frequency of both chromosome-type and chromatid-type aberrations, which could potentially lead to mis-segregation. The role of chromosome aberrations and other factors, including chromosome lagging, premature chromatid separation, and centrosome malfunctioning on chromosome mis-segregation in irs1 and irs1SF cells have been investigated. In particular, the linkage of DNA repair proteins with centrosomes suggests a key role for the centrosome in controlling cellular repair processes.

Role of mammalian RAD51L2 (RAD51C) in recombination and genetic stability.

The highly conserved RAD51 protein has a central role in homologous recombination. Five novel RAD51-like genes have been identified in mammalian cells, but little is known about their functions. A DNA damage-sensitive hamster cell line, irs3, was found to have a mutation in the RAD51L2 gene and an undetectable level of RAD51L2 protein. Resistance of irs3 to DNA-damaging agents was significantly increased by expression of the human RAD51L2 gene, but not by other RAD51-like genes or RAD51 itself. Consistent with a role for RAD51L2 in homologous recombination, irs3 cells show a reduction in sister chromatid exchange, an increase in isochromatid breaks, and a decrease in damage-dependent RAD51 focus formation compared with wild type cells. As recently demonstrated for human cells, we show that RAD51L2 forms part of two separate complexes of hamster RAD51-like proteins. Strikingly, neither complex of RAD51-like proteins is formed in irs3 cells. Our results demonstrate that RAD51L2 has a key role in mammalian RAD51-dependent processes, contingent on the formation of protein complexes involved in homologous recombination repair.