BRCT domain (BRCA1 C-terminal domain) is an important signaling and protein targeting motif in DNA damage response system. BRCT domain, which mainly occur as singleton (single BRCT) or tandem pair (double BRCT), contains a phosphate binding pocket that can bind the phosphate from either DNA end or phosphorylated protein motif. There are also BRCT domains that can bind proteins in phospho-independent manner. By using increasing amount of information on crystal structure and function, we analyzed the functional evolution of BRCT domain and predicted its new functional mechanism. Firstly, we performed database search, phylogeny reconstruction, and phosphate binding pocket comparison to analyze the functional evolution of BRCT domain. We identified new BRCT domain-containing proteins in bacteria and eukaryotes, and found that the number of BRCT containing proteins per genome is correlated with genome complexity. The phylogeny analyses revealed that there are two groups of single BRCT domain (sGroup I and sGroup II) and double BRCT domain (dGroup I and dGroup II). These four BRCT groups showed differences in the phosphate binding pocket. In eukaryotes, the evolution of BRCT domain can be divided into three phases. In the first phase, sGroup I proteins derived the bacteria BRCT fold with the phosphate binding pocket that can bind the phosphate of nicked DNA. In the second phase, the phosphate binding pocket changed from DNA binding type to phosphorylated protein motif binding type in sGroup II. The tandem duplication of sGroup II BRCT domain gave birth to double BRCT domain, from which two structurally and functionally distinct groups were evolved. Both sGroup I and sGroup II BRCT domains originated in third phase lost the phosphate binding pocket and many evolved protein binding sites. Many dGroup I members were evolved in this phase but few dGroup II members were observed. In addition, BRCT domain can evolve and function with three BRCT domains (triple BRCT) as a unit, and the phosphate binding pocket in triple BRCT can bind phosphorylated protein motif. The results further suggested that the BRCT domain expansion and functional change in eukaryote may be driven by the evolution of the DNA damage response system. Secondly, we predicted the phosphorylated protein binding mechanism of XRCC1 BRCT1, PTIP BRCT4, ECT2 BRCT1 and TopBP1 BRCT1. The structural conservation and electrostatic surface calculation showed that conserved grooves with positive potential, which are common among phosphate binding pocket-containing BRCT domains, may be the functional sites of these four BRCT domains. The two sides of grooves are composed of positively charged or hydrophilic residues while the bottoms are composed of hydrophobic residues, suggesting that these grooves may bind to proteins mainly through electrostatic and hydrophobic interactions. The grooves in the four BRCT domains, each of which mainly locates in one BRCT domain, are different in shape and charge distribution indicating that the binding specificity is mainly determined by one BRCT domain. The groove is centered at the phosphate binding pocket implying that the groove can bind both the N-terminal and C-terminal residues of the phosphate containing residue.
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