Thiopurine-induced mitotic catastrophe in Rad51d-deficient mammalian cells.

Thiopurines are part of a clinical regimen used for the treatment of autoimmune disorders and childhood acute lymphoblastic leukemia. However, despite these successes, there are also unintended consequences such as therapy-induced cancer in long-term survivors. Therefore, a better understanding of cellular responses to thiopurines will lead to improved and personalized treatment strategies. RAD51D is an important component of homologous recombination (HR), and our previous work established that mammalian cells defective for RAD51D are more sensitive to the thiopurine 6-thioguanine (6TG) and have dramatically increased numbers of multinucleated cells and chromosome instability. 6TG is capable of being incorporated into telomeres, and interestingly, RAD51D contributes to telomere maintenance, although the precise function of RAD51D at the telomeres remains unclear. We sought here to investigate: (1) the activity of RAD51D at telomeres, (2) the contribution of RAD51D to protect against 6TG-induced telomere damage, and (3) the fates of Rad51d-deficient cells following 6TG treatment. These results demonstrate that RAD51D is required for maintaining the telomeric 3' overhangs. As measured by γ-H2AX induction and foci formation, 6TG induced DNA damage in Rad51d-proficient and Rad51d-deficient cells. However, the extent of γ-H2AX telomere localization following 6TG treatment was higher in Rad51d-deficient cells than in Rad51d-proficient cells. Using live-cell imaging of 6TG-treated Rad51d-deficient cells, two predominant forms of mitotic catastrophe were found to contribute to the formation of multinucleated cells, failed division and restitution. Collectively, these findings provide a unique window into the role of the RAD51D HR protein during thiopurine induction of mitotic catastrophe. Environ. Mol. Mutagen. 59:38-48, 2018. © 2017 Wiley Periodicals, Inc.

Extensive chromosomal instability in Rad51d-deficient mouse cells.

Homologous recombination is a double-strand break repair pathway required for resistance to DNA damage and maintaining genomic integrity. In mitotically dividing vertebrate cells, the primary proteins involved in homologous recombination repair are RAD51 and the five RAD51 paralogs, RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3. In the absence of Rad51d, human and mouse cells fail to proliferate, and mice defective for Rad51d die before birth, likely as a result of genomic instability and p53 activation. Here, we report that a p53 deletion is sufficient to extend the life span of Rad51d-deficient embryos by up to 6 days and rescue the cell lethal phenotype. The Rad51d-/- Trp53-/- mouse embryo-derived fibroblasts were sensitive to DNA-damaging agents, particularly interstrand cross-links, and exhibited extensive chromosome instability including aneuploidy, chromosome fragments, deletions, and complex rearrangements. Additionally, loss of Rad51d resulted in increased centrosome fragmentation and reduced levels of radiation-induced RAD51-focus formation. Spontaneous frequencies of sister chromatid exchange were not affected by the absence of Rad51d, but sister chromatid exchange frequencies did fail to be induced upon challenge with the DNA cross-linking agent mitomycin C. These findings support a crucial role for mammalian RAD51D in normal development, recombination, and maintaining mammalian genome stability.

The ATPase motif in RAD51D is required for resistance to DNA interstrand crosslinking agents and interaction with RAD51C.

Homologous recombination (HR) is a mechanism for repairing DNA interstrand crosslinks and double-strand breaks. In mammals, HR requires the activities of the RAD51 family (RAD51, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1), each of which contains conserved ATP binding sequences (Walker Motifs A and B). RAD51D is a DNA-stimulated ATPase that interacts directly with RAD51C and XRCC2. To test the hypothesis that ATP binding and hydrolysis by RAD51D are required for the repair of interstrand crosslinks, site-directed mutations in Walker Motif A were generated, and complementation studies were performed in Rad51d-deficient mouse embryonic fibroblasts. The K113R and K113A mutants demonstrated a respective 96 and 83% decrease in repair capacity relative to wild-type. Further examination of these mutants, by yeast two-hybrid analyses, revealed an 8-fold reduction in the ability to associate with RAD51C whereas interaction with XRCC2 was retained at a level similar to the S111T control. These cell-based studies are the first evidence that ATP binding and hydrolysis by RAD51D are required for efficient HR repair of DNA interstrand crosslinks.

Telomere maintenance requires the RAD51D recombination/repair protein.

The five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are required in mammalian cells for normal levels of genetic recombination and resistance to DNA-damaging agents. We report here that RAD51D is also involved in telomere maintenance. Using immunofluorescence labeling, electron microscopy, and chromatin immunoprecipitation assays, RAD51D was shown to localize to the telomeres of both meiotic and somatic cells. Telomerase-positive Rad51d(-/-) Trp53(-/-) primary mouse embryonic fibroblasts (MEFs) exhibited telomeric DNA repeat shortening compared to Trp53(-/-) or wild-type MEFs. Moreover, elevated levels of chromosomal aberrations were detected, including telomeric end-to-end fusions, a signature of telomere dysfunction. Inhibition of RAD51D synthesis in telomerase-negative immortalized human cells by siRNA also resulted in telomere erosion and chromosome fusion. We conclude that RAD51D plays a dual cellular role in both the repair of DNA double-strand breaks and telomere protection against attrition and fusion.