p53 dependent S checkpoint in early mouse embryos and radiation induction of genomic instability

在受照于精子阶段的雄性小鼠所生的F1小鼠中，发现母系衍生的Ms6hM小卫星基因座和粉眼不稳定基因的突变水平升高。这些突变证明了由于基因组不稳定而导致的非靶向和延迟重组突变的操作，这种不稳定是在小鼠发育早期由受辐射的精子引起的DNA辐射损伤引起的。为了阐明基因组不稳定性的分子机制，我们开展了精子辐照小鼠胚胎损伤反应的研究。受精卵与辐照精子受精后，显微注射到受精卵中的p53报告基因在转录水平上被激活。这种原核串扰被发现抑制了精子照射受精卵中雌性原核和雄性原核的DNA合成。在P53-/-受精卵中没有观察到这种DNA合成的抑制，而经辐照的P53-/-受精卵被显微注射P53蛋白后恢复，提示了一条新的S检查点途径。将突变的P53蛋白显微注射到辐照的P53-/-受精卵中，结果表明，这个依赖于P53的S检查点需要P53的DNA结合结构域，但需要反式激活结构域。对原代小鼠的依赖于P53的S检查点进行了分析，发现其工作剂量低于2GY。此外，通过ID U和CldU双标记细胞的DNA纤维伸长分析表明，依赖于P53的S检查点通过减缓复制分叉进程而抑制DNA合成。这种复制的减慢与姐妹染色单体之间较高的重组率有关，这可以解释小卫星的非靶向重组。在粉色眼睛不稳定的等位基因上观察到的延迟重组需要另一种损伤记忆的机制，对于这一机制，还需要更多的研究。

Elevated levels of mutations were found at the maternally derived alleles of the Ms6hm minisatellite locus and the pink-eyed unstable locus in F1 mice born to male mice irradiated at the spermatozoa stage. These mutations demonstrated the operation of untargeted and delayed recombinational mutations due to genomic instability which was induced during early mouse development by radiation DNA damage introduced by the irradiated sperm. In order to elucidate the molecular mechanism of genomic instability, we have undertaken studies of damage response in sperm irradiated mouse embryos.A p53 responsible reporter plasmid microinjected into zygotes was transcriptionally activated when they zygotes were fertilized with irradiated sperm. This pronuclear cross-talk was found to suppress DNA synthesis of female pronucleus as well as of male pronucleus in sperm irradiated zygotes. This suppression of DNA synthesis was not observed in p53-/-zygotes which was restored when the sperm irradiated p53-/-zygotes were microinjected with p53 protein, suggesting a novel pathway of S checkpoints. Microinjection of mutant p53 proteins into sperm irradiated p53-/-zygotes demonstrated that this p53 dependent S checkpoint required DNA binding domain of p53,but the transactivation domain.The p53 dependent S checkpoint was analyzed in primary mouse and found operate at doses below 2 Gy. In addition, analyses of DNA fiber elongation by double labeling the cells with Id U and Cld U showedthatthe p53 dependent S checkpoint suppressed DNA synthesis by slowing down of replication fork progression. This slowing down of replication was associated with a higher rate of recombination between sister chromatids which can explain the untargeted recombination of minisatellite. Delayed recombination observed at the pink-eyed unstable allele requires yet another mechanism of damage memory for which much more to be studied yet.