Abstract
Phosphorylation of p53 by ATM and ATR is important for its stabilization and activation after DNA damage. Caffeine is known to inhibit ATM and ATR kinase activities, whereas wortmannin represses that of ATM. Both reagents dramatically abolished the suppression of DNA synthesis in sperm-irradiated zygotes, whereas they had no effect on the unirradiated zygotes. These suggest that S checkpoint in mouse zygotes is mainly controlled by the ATM pathway. Microinjection p53 mutant, which lacks the DNA binding capacity, interfered with the suppression of DNA synthesis in sperm-irradiated zygotes. Inhibiting transcription with α-amanitin, an inhibitor of RNA polymerase II, had no effect on the suppression of DNA synthesis in sperm-irradiated mouse zygotes. The above data suggest that p53 suppresses DNA synthesis in a transactivation-independent manner. DNA binding of p53 might be important in S checkpoint of sperm-irradiated mouse zygotes.To investigate the role of p53 in sperm-irradiated mouse embryo, they were immunostained with Rad51 which is required for recombination repair. Rad51 was stained in mouse zygotes irradiated directly but not un-irradiated. Surprisingly, Rad 51 was also stained in male and female pronuclei of sperm-irradiated mouse zygotes. m5s cells were transiently transfected with GFP-p53 wt or GFP-p53 R273H. DNA binding domain of p53 associates with Rad 51 in vivo. These suggest that p53 could recruit Rad51 to replication fork for repair.
