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.
During the formation of sperm and eggs, chromosomes must segregate accurately. However, approximately 10% of clinically reported pregnancies are chromosomally abnormal, resulting in pregnancy loss or genetic defects such as Down syndrome. Very little is known about the genetic failures that cause these chromosomal abnormalities in sex cells;however, we will significantly enhance our knowledge of this phenomenon by achieving the goals set within this proposal.