MERIT40 (MEdiator of RAP80 Interaction and Targeting 40) is a novel associate of the BRCA1-complex and plays an essential role in DNA damage repair. It is the least characterized protein of BRCA1-complex and mainly responsible for maintaining the complex integrity. However, its structural and functional aspects of regulating the complex stability still remain elusive. Here, we carried out a comprehensive examination of MERIT40 biophysical properties and identified its novel interacting partner which would help to understand its role in BRCA1-complex. The recombinant protein was purified by affinity chromatography and unfolding pathway was determined using spectroscopic and calorimetric methods. Molecular model was generated using combinatorial approaches of modeling, and monomer-monomer docking was carried out to identify dimeric interface. Disordered region of MERIT40 was hatchet using trypsin and chymotrypsin to illustrate the existence of stable domain whose function was speculated through DALI search. Our findings suggest that MERIT40 forms a dimer in a concentration-independent manner. Its central region shows remarkable stability towards the protease digestion and has structural similarity with vWA-like region, a domain mainly present in complement activation factors. MERIT40 undergoes a three-state unfolding transition pathway with a dimeric intermediate. It interacts with adaptor molecule of BRCA1-complex, called ABRAXAS, thus help in extending the bridging interaction among various members which further stabilizes the whole complex. The results presented in this paper provide first-hand information on structural and folding behavior of MERIT40. These findings will help in elucidating the role of protein-protein interactions in stabilization of BRCA1-complex.

Receptor Associated Protein 80 (RAP80) is a member of RAP80-BRCA1-CCDC98 complex family and helps in its recruitment to the DNA damage site for effective homologous recombination repair. It encompasses two tandem UIMs (UIM1 and UIM2) motif at its N-terminus, which interact with K-63 linked polyubiquitin chain(s) on H2AX and thereby assemble the RAP80-BRCA1 complex at the damage site. Nevertheless, how RAP80 helps in the structural integrity of BRCA1 complex is still elusive. Considering the role of RAP80 in the recruitment of BRCA1 complex at the DNA damage site, we attempted to explore the molecular mechanism associated with RAP80 and mutation that causes chromosomal aberrations due to its loss of function. There is a significant loss in structural characteristics of RAP80 ΔE81, which impairs its binding affinity with the polyubiquitin chain. This leads to the defective recruitment of RAP80 and BRCA1 complex at the DNA damage site. The results presented here are very useful in understanding the cause of various repair defects (chromosomal aberration) that arise due to this mutation. Comparative study of wild type and ΔE81 could be helpful in designing the small molecules that can potentially compensate the deleterious effect(s) of ΔE81 and hence useful for therapeutic application.