Abstract
The BRCT domain of BARD1 (BARD1 BRCT) is involved in many cellular processes such as DNA damage repair (DDR) and cell-cycle checkpoint regulation. BARD1 BRCT performs tumor suppressor function by recruiting BRCA1 at DNA damage site via interactions with other DNA damage repair (DDR) proteins. Considering the importance of the BRCT domain in genomic integrity, we decided to evaluate reported mutations of BARD1 BRCT Cys645Arg, Val695Leu, and Ser761Asn for their pathogenicity. To explore the effect of the mutation on the structure and function, BARD1 BRCT wild-type proteins and the mutant proteins were studied using different biochemical, biophysical and in silico techniques. Comparative fluorescence, circular dichroism (CD) spectroscopy and limited proteolysis studies demonstrate the well-folded structural conformation of wild-type and mutant proteins. However, thermal and chemical denaturation studies revealed similarity in the folding pattern of BARD1 BRCT wild-type and Cys645Arg mutant proteins, whereas there was a significant loss in the thermodynamic stability of Val695Leu and Ser761Asn mutants. Molecular dynamics (MD) simulation studies on wild-type and mutant protein structures indicate the loss in structural integrity of mutants compared with the wild-type protein.
Highlights
The BARD1 BRCT Cys645Arg, Val695Leu, and Ser761Asn mutations are identi ed as cancer predisposing in nature (Fig. 1A, Electronic supplementary information (ESI)†), and signi cantly affect the tumor-suppressor functions of the BARD1.15 The DDG value predicted by Imutant 3.0 is tabulated in Table 1 (ESI†)
It is observed that DDG value predicted for BARD1 BRCT Cys645Arg mutant protein is À0.29 kcal molÀ1, which is smaller than the set threshold of À0.5 kcal molÀ1
Align-GVGD classi es BARD1 BRCT Cys645Arg mutant protein as most likely to be pathogenic and scores this protein as class 65, while Ser761Asn is scored as class 45 and Val695Leu is scored as class 25, classifying as less pathogenic (Table 1)
Summary
BRCT repeat domains are present in different DNA repair proteins, such as MDC1, BRCA1, BARD1, XRCC1, and 53BP1, and dynamically participate in DNA damage repair mechanism.[18,19,20,21,22,23,24] BARD1 BRCT has two conserved binding pockets, P1 (hydrophilic pocket) and P2 (hydrophobic pocket), for interaction with DNA damage repair protein in a phosphodependent manner.[25]. 32) (prediction of protein stability changes upon single point mutation), and SNP&GO33 (single amino acid polymorphisms using GO terms) have been used for the prediction of pathogenicity of mutations. To understand the abrogative effects of mutations, a combinatorial approach of biochemical, biophysical and in silico techniques were employed. This study will help in understanding the role of BARD1 BRCT in DNA damage repair and future development of small molecule inhibitors, which can modulate or neutralize the pathogenic effects of mutations
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
