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.
The proposed research is relevant to public health because the recognition of protein binding modules has been implicated in the susceptibility to many forms of cancer. Thus the proposed research is relevant to the part of NIH's mission that aims to foster fundamental creative discoveries, innovative research strategies as a basis for protecting and improving health.