Analysis of meiotic DNAdamage checkpoint

由于没有建立有效的分析方法，人类减数分裂的分子机制仍然是未知的。我们研究了裂殖酵母减数分裂过程中DNA损伤的检查点系统，在营养细胞中，检查点Rad（Rad1，Rad3等）响应于羟基脲（HU）的Chk1的依赖性活化将细胞周期阻滞在G2。但在减数分裂过程中，Chk 1依赖的对HU的反应减弱。为了研究减数分裂DNA损伤检查点系统的存在，在减数分裂早期用烷化剂甲基甲烷磺酸盐（MMS; 0.01%）处理rad1，chk1，cds1突变体。这些突变体以与野生型相似的速率经历异常的染色体分离、延迟和异常的减数分裂。与营养细胞相比，在具有MMS的减数分裂cds1细胞中，在免疫印迹中观察到Chk1蛋白的细微磷酸化。Cdc2-Tyr15在减数分裂S期也被脱磷酸化。本研究中检测点突变体对MMS的DNA损伤反应表明裂殖酵母减数分裂中的DNA损伤检测点被减弱或不存在。最近，其他研究小组已经证明自发的S期损伤是通过激活重组而修复的，而没有检测点停滞。尚未显示检查点畸变是否导致人类减数分裂异常。在人类中已经发现了一些与DNA重组相关的基因，因此研究这些基因对了解减数分裂的分子机制具有重要意义。

Molecular mechanism in human meiosis is still unknown as no effective methods are established to analyze. We examined checkpoint system to DNA damage during fission yeast meiosis.In vegetative cdsl cells, checkpoint Rad (Rad1, Rad3 etc.) dependent activation of Chk1 in response to hydroxyurea(HU) arrest the cell cycle in G2. But Chk1 dependent response to HU during meiosis is attenuated. To investigate the existence of meiotic DNA damage checkpoint system, rad1, chk1, cds1 mutants are treated with alkylating agent methylmethane sulfonate(MMS; 0.01%) during early meiosis. These mutants undergo aberrant chromosomal segregation, delayed and abnormal meiotic divisions at s similar rate to wild type. In meiotic cds1 cells with MMS, subtle phosphorylation in Chk1 protein is observed in immunoblot, compared to the vegetative cells. Cdc2-Tyr15 in meiotic S phase is also dephosphorylated. The DNA damage response of checkpoint mutants to MMS in this study suggests DNA damage checkpoint in fission yeast meiosis is attenuated or not in existence.Recently, other groups have demonstrated that spontaneous S phase damage is repaired by activating recombination without checkpoint arrest. It is not shown whether the checkpoint aberration causes meiotic abnormality in human. As some genes related to the DNA recombination have already identified in human, it would be significant to examine the genes to know the molecular mechanism in meiosis.