Elucidating binding modes of BRCT-modules

描述（由申请人提供）：我们对磷酸丝氨酸结合模块如何通过BRCT结构域实现多蛋白DNA修复复合物组装的机制的理解存在根本性的空白。忽视这一差距是一个重要的问题，因为在解决这一问题之前，对DNA修复调节缺陷如何可能导致癌症的理解仍将是模糊的。长期目标是了解全长PTIP如何调节DNA损伤反应途径。这一特殊应用的目的是提供PTIP-(BRCT)4相互作用如何帮助调节DNA修复的分子描述。核心假设是（BRCT）4能够识别最初确定的更广泛的磷酸肽序列基序，并且它可以介导与其他含有BRCT结构域的蛋白质的相互作用。这一假设是根据申请人实验室获得的初步数据制定的。这项研究的基本原理是，一旦知道PTIP相互作用如何传递DNA损伤信号来调节肿瘤细胞中的DNA修复，我们就可以确定破坏这些相互作用的药物，从而使它们对化疗和放疗更敏感。

DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of how mechanisms of phosphoserine binding modules by BRCT domains enable the assembly of multiprotein DNA repair complexes. Overlooking this gap is an important problem because, until it is solved, understanding how defects in the regulation of DNA repair can potentially cause cancer will remain obscure. The long-term goal is to understand how full-length PTIP regulates the DNA damage response pathway. The objective of this particular application is to provide a molecular description of how PTIP-(BRCT)4 interactions help to regulate DNA repair. The central hypothesis is that (BRCT)4, is able to recognize a broader spectrum of phosphopeptide sequence motifs, that were initially determined, and it can mediate interactions with other BRCT-domain containing proteins. This hypothesis has been formulated based on preliminary data obtained in the applicant's lab. The rationale for the proposed research is that, once it is known how PTIP interactions relay DNA damage signals to regulate DNA repair in tumor cells, we can identify agents that disrupt these interactions thereby making them more sensitive to chemo- and irradiation therapies. This hypothesis will be tested by investigation of three aims: 1) determination of the X-ray crystal structure of the tandem pairs of C-terminal BRCT domains; 2) determination of the structural basis of substrate specificity of the tandem pair of BRCT domains; 3) determination of the mechanism of substrate preference of the tandem pairs of BRCT domains. Within the first aim, native crystals diffract to 2.8 A and the heavy atom derivative diffracts to 3.2 A. Automated software programs will be used to solve the structure. Within the second aim, we have purified sufficient amounts of protein for biochemical and structural investigations and will begin crystallization screening and direct peptide binding experiments using surface Plasmon resonance (SPR). Lastly, within the third aim, peptide arrays will be conducted to identify novel sequences recognized by (BRCT)4, and confirmed with SPR. This approach is innovative, in my opinion, because it will be the first comprehensive study in the field focused on the importance of four consecutive BRCT domains. The proposed research is significant, because it will elucidate the differences in binding mechanisms among BRCT domains, and fundamentally expand our understanding of protein recognition with BRCT-domain containing proteins. Ultimately, this knowledge can identify agents that disrupt BRCT domain interactions and thereby make tumor cells more sensitive to killing by chemo- and irradiation therapies.