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.

The Lys63-deubiquitylating Enzyme BRCC36 Limits DNA Break Processing and Repair

“…A more recent study also revealed that an additional phosphorylation event in the vicinity of pSPXF (serine 404) further stabilizes the Abraxas-BRCA1 interaction by formation of a stable dimer (16). Other components of the BRCA1-A complex, including Merit40 and BRE, are also key in maintaining the integrity of the macromolecular protein complex (6,9,17,18) and for BRCA1 accumulation at DSB sites. Together, these data underscore critically important roles of each of the subunits that make up the protein assembly.…”

Multimodal approach to explore the pathogenicity of BARD1, ARG 658 CYS, and ILE 738 VAL mutants

“…the folded (N) to intermediate (I), and intermediate to unfolded (U) transitions of the curve.Further thermodynamic constraints, analysis and curve fitting were performed(Pace & Shaw 2000;Vikrant et al 2014).…”

Biophysical evaluation to categorize pathogenicity of cancer-predisposing mutations identified in the BARD1 BRCT domain

“…3A). The T m 's values calculated for BARD1 BRCT wild-type, Cys645Arg, Val695Leu and Ser761Asn assuming a two-state transition model 34,38,39 were 47.7 AE 0.85 C, 47.6 AE 0.65 C, 42.3 AE 0.78 C, and 41.1 AE 0.42 C, respectively. DG N-D calculated for BARD1 BRCT wildtype, Cys645Arg, Val695Leu, and Ser761Asn mutant proteins were 9.8 AE 0.39 kcal mol À1 , 9.6 AE 0.11 kcal mol À1 , 7.4 AE 0.69 kcal mol À1 and 7.1 AE 0.12 kcal mol À1 , respectively (Fig.…”

“…In chemical denaturation, a three-state model (folded (N), intermediate (I) and unfolded (U)) was used for curve tting. 34,38,39 Furthermore, circular dichroism spectra of BARD1 BRCT wild-type and mutant proteins were recorded using sealed quartz cuvettes of 1 mm path length using a JASCO J-815 spectropolarimeter (Jasco, Easton, MD). BARD1 BRCT wild-type and mutant proteins at concentrations of 10 mM and 40 mM were scanned in the far-UV (l ¼ 200-240 nm) and near-UV range (l ¼ 350-250 nm), respectively.…”