The SMC5/6 Complex - DNA Damage Response Regulation Ensures Meiotic Fidelity

描述(由申请人提供)：我们知道在减数分裂过程中DNA损伤反应受到干扰会导致不孕、妊娠丢失或遗传缺陷。然而，我们对减数分裂过程中DNA损伤反应的调控知之甚少。我的初步数据表明，在减数分裂细胞周期进程中，SMC5/6复合体是介导DNA损伤反应所必需的。由于SMC5/6复合体是细胞存活所必需的，因此还没有在哺乳动物身上进行体内实验，我们对SMC5/6在哺乳动物中的功能的了解也是有限的。我正在创造一种生殖细胞特异性的Smc5突变，并突变睾丸特有的SMC5/6基因Eid3。这些突变体将使我能够对SMC5/6复合体在减数分裂中的功能进行第一次全面的研究(Aim1)。EID3是SMC5/6复合体中睾丸特异的Kleisin亚基。然而，体细胞Kleisin SMC5/6亚单位NSE4也在睾丸中表达。因此，睾丸内存在两种SMC5/6复合体，SMC5/6NSE4和SMC5/6EID3。我将确定这两个复合体之间的相似和不同之处(目标2)。我已经证实SMC5/6复合体与BAT3-EP300复合体相互作用。BAT3-EP300复合体是激活TrP53介导的DNA损伤反应所必需的。根据SMC5/6与BAT3-EP300复合体之间形成的蛋白质相互作用网络，SMC5/6通过调节Trp53乙酰化而成为DNA损伤反应途径的关键组成部分。在目标3中，我将对SMC5/6与BAT3-EP300的相互作用进行详细的评估，并对EID3对BAT3-EP300介导的Trp53乙酰化的拮抗作用进行生化分析。我对K99/R00奖的假设是，SMC5/6作为一个修复/监视复合体，协调DNA修复和Trp53介导的DNA损伤反应。这一功能确保了减数分裂前期到中期I转换的保真度。

DESCRIPTION (provided by applicant): We know a perturbed DNA damage response during meiosis will result in infertility, pregnancy loss or genetic defects. However, we know very little about the regulation of the DNA damage response during meiosis. My preliminary data implies that the SMC5/6 complex is required to mediate the DNA damage response during meiotic cell cycle progression. Because the SMC5/6 complex is essential for cell viability, in vivo experiments in mammals have not been performed and our understanding of the function of SMC5/6 in mammals is limited. I am creating a germ cell-specific mutation of Smc5 and mutating the testis specific SMC5/6 gene Eid3. These mutants will enable me to perform the first comprehensive studies of SMC5/6 complex function in meiosis (Aim1). EID3 is a testis-specific kleisin subunit of the SMC5/6 complex. However, the somatic cell kleisin SMC5/6 subunit, NSE4, is also expressed within the testis. Therefore, there are two SMC5/6 complexes present within the testis, SMC5/6NSE4 and SMC5/6EID3. I will determine the similarities and differences between the two complexes (Aim 2). I have confirmed that the SMC5/6 complex interacts with the BAT3-EP300 complex. The BAT3-EP300 complex is required for the activation of a TRP53-mediated DNA damage response. According to the protein interaction network developed between the SMC5/6 and the BAT3-EP300 complexes, SMC5/6 emerges as a key component of the DNA damage response pathway, by regulating TRP53 acetylation. In Aim 3, I will conduct a detailed assessment of the interaction between SMC5/6 and BAT3-EP300, together with biochemical analysis of the antagonistic function of EID3 on BAT3-EP300-mediated TRP53 acetylation. I approach the K99/R00 award with the hypothesis that SMC5/6 acts as a repair/surveillance complex coordinating DNA repair and the TRP53-mediated DNA damage response. This function ensures fidelity at the prophase to metaphase I transition of meiosis.