Elucidating binding modes of BRCT-modules

描述(由申请人提供)：在我们对BRCT结构域的磷酸丝氨酸结合模块如何使多蛋白DNA修复复合体组装的机制的理解上存在着根本的差距。忽视这一差距是一个重要的问题，因为在这个问题得到解决之前，了解DNA修复调控缺陷如何可能导致癌症仍将是一个模糊的问题。长期目标是了解全长PTIP是如何调节DNA损伤反应途径的。这一特殊应用的目的是提供关于PTIP-(BRCT)4相互作用如何帮助调节DNA修复的分子描述。中心假设是(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。这一假设是基于申请人实验室获得的初步数据提出的。这项拟议研究的基本原理是，一旦了解了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，我们就可以识别破坏这些相互作用的药物，从而使它们对化疗和放射治疗更加敏感。 这一假说将通过三个目的的研究来验证：1)确定C-末端BRCT结构域串联对的X射线晶体结构；2)确定BRCT结构域串联对底物特异性的结构基础；3)确定BRCT结构域串联对底物偏好的机制。在第一个目标内，自然晶体衍射率为2.8A，重原子导数衍射率为3.2A，将使用自动软件程序来求解结构。在第二个目标中，我们已经提纯了足够数量的蛋白质用于生化和结构研究，并将开始使用表面等离子体共振(SPR)进行结晶筛选和直接肽结合实验。最后，在第三个目标中，将进行肽阵列来识别(BRCT)4识别的新序列，并通过SPR进行确认。在我看来，这种方法是创新的，因为这将是该领域第一次全面研究连续四个金砖四国领域的重要性。这项研究意义重大，因为它将阐明BRCT结构域之间结合机制的差异，并从根本上扩大我们对含有BRCT结构域的蛋白质识别的理解。最终，这一知识可以确定破坏BRCT结构域相互作用的药物，从而使肿瘤细胞对化疗和放射治疗的杀伤更加敏感。

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.