Structural and functional characterization of the MERIT40 to understand its role in DNA repair

Abstract

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.

Keywords:

• genomic stability
• MERIT40
• BRCA1
• RAP80
• DNA damage repair (DDR)

Notes

Funding for this study was supported by DBT (No. BT/PR10765/BRB/664/2008 and BT/PR12565/BID/07/303/2009), Seed in Air grant from TMC. We thank DBT-BTIS facility at ACTREC for providing the necessary software for this study. Mr Vikrant thanks CSIR for fellowship (09/513 (0072)/2008-EMR-I). We thank Dr Hosur and Dr Lata (BARC) for providing DSC facility and data analysis.

The supplementary material for this paper is available online at http://dx.doi.[10.1080/07391102.2013.843473].