Activities of wildtype and mutant p53 in suppression of homologous recombination as measured by a retroviral vector system.
DNA repair of double strand breaks, interstrand DNA cross-links, and other types of DNA damage utilizes the processes of homologous recombination and non-homologous end joining to repair the damage. Aberrant homologous recombination is likely to be responsible for a significant fraction of chromosomal deletions, duplications, and translocations that are observed in cancer cells. To facilitate measurement of homologous recombination frequencies in normal cells, mutant cells, and cancer cells, we have developed a high titer retroviral vector containing tandem repeats of mutant versions of a GFP-Zeocin resistance fusion gene and an intact neomycin resistance marker. Recombination between the tandem repeats regenerates a functional GFP-Zeo(R) marker that can be easily scored. This retroviral vector was used to assess homologous recombination frequencies in human cancer cells and rodent fibroblasts with differing dosages of wild type or mutant p53. Absence of wild type p53 stimulated spontaneous and ionizing radiation-induced homologous recombination, confirming previous studies. Moreover, p53(+/-) mouse fibroblasts show elevated levels of homologous recombination compared to their p53(+/+) counterparts following retroviral vector infection, indicating that p53 is haploinsufficient for suppression of homologous recombination. Transfection of vector-containing p53 null Saos-2 cells with various human cancer-associated p53 mutants revealed that these altered p53 proteins retain some recombination suppression function despite being totally inactive for transcriptional transactivation. The retroviral vector utilized in these studies may be useful in performing recombination assays on a wide array of cell types, including those not readily transfected by normal vectors.
Lu
Xiongbin
X
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.
Lozano
Guillermina
G
Donehower
Lawrence A
LA
D004260:DNA Repair; D005819:Genetic Markers; D005822:Genetic Vectors; D006801:Humans; D020125:Mutation, Missense; D011839:Radiation, Ionizing; D011995:Recombination, Genetic / Q000528:radiation effects; D012190:Retroviridae; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D005819:Genetic Markers; D016159:Tumor Suppressor Protein p53