Loss of wild-type p53 function is responsible for upregulated homologous recombination in immortal rodent fibroblasts

Purpose; A correlation between mutations in the tumour suppressor gene p53 and high rates of homologous recombination were previously found in immorta l rodent fibroblasts. In the current study, direct evidence was sought that loss of p53 function is mainly responsible for upregulated levels of homol ogous recombination. Materials and methods: Homologous recombination was assessed in vitro using DNA plasmid substrates that stably integrated into the genome of mouse and rat embryonic fibroblasts. Results: Primary fibroblasts with wild-type p53 displayed a recombination r ate of about 1 x 10(-4). This number increased by 33- to 93-fold after spon taneous cellular immortalization, accompanied by loss of p53 function. To e xclude potential bias from other gene mutations, wild-type p53 was experime ntally disrupted in primary fibroblasts leading to an increase in recombina tion by one order of magnitude. Conversely, re-introduction of wild-type p5 3 into p53-null immortal cells reconstituted suppressed recombination rates . Finally, early-passage fibroblast cultures from p53-knock-out mice showed elevated recombination rates, which did not increase further following imm ortalization. Conclusions: Loss of wild-type P53 is the major genetic determinant of incr eased homologous recombination frequencies in immortal rodent fibroblasts. Cellular P53 status will be an important factor to consider when performing functional analysis of the increasing number of mammalian proteins that ar e found to be involved in homologous recombination.