BRC is a signature motif and defining element of the BRCA2 family of proteins. After the gene for human BRCA2, the founding member of the family, was initially cloned, it was found to have no sequence similarity to any known protein or domain. The first clue to elucidating its function was recognition of a tandemly arranged array of closely related sequences—the BRC repeats [1], [2]. These were found to associate with RAD51 protein, a central component of the homologous recombination (HR) system [3], [4], establishing a connection between BRCA2 and DNA repair by HR and leading ultimately to recognition of BRCA2 itself as a pivotal factor in HR. Besides the BRC repeats, BRCA2 harbors within its C-terminal region the DBD, a domain composed of a helix-rich region and one to several tandem OB folds that bind single stranded DNA as well as the small acidic intrinsically unstructured protein DSS1 [5]. The BRC repeats together with the DBD comprise structural identifiers that define and distinguish the BRCA2 protein family [6]. Remarkably, of these two types of structural constituents, the BRC repeats are essential for BRCA2 function, while the DBD is expendable under certain conditions [7], [8], [9].

The BRC is an evolutionarily conserved motif of about 35 amino acids (aa) that can be found in multiple copies in metazoans, often arranged tandemly in clusters distributed at intervals and almost always located proximal in the protein sequence relative to the DBD. In human BRCA2 there are eight nonidentical BRC repeats spaced unevenly but localized to the medial region of the protein. From the structure of human BRC4 peptide crystallized in complex with a polypeptide fragment comprising the catalytic core domain of RAD51, it was determined that interaction of BRC4 peptide (HsBRC4 in discussion below) with RAD51 extends along a stretch of 28 residues in continuous contact with RAD51 from a β-hairpin at the proximal end of the BRC through a spacer region to an α-helix at the distal end [10]. Hydrophobic as well as polar residues in this sequence form multiple, independent points of contact along the surface of RAD51. Tetrameric clusters of hydrophobic residues at either end comprise two modules that anchor into distinct pockets in RAD51 [11]. The proximal tetramer module contains a highly conserved FxxA sequence with the F and A residues buried in the RAD51 interface, while the distal tetramer contains a consensus ϕϕx-/Q sequence (ϕ = hydrophobic, - = acidic) with ϕ residues buried in the second hydrophobic pocket. These proximal and distal tetrameric modules together with hydrophobic and polar residues in the intervening spacer region form a strip that lies across the RAD51 surface creating an orthogonal clamp.

Structural studies with the BRC4-RAD51 complex revealed that the FxxA sequence within the context of the β-hairpin mimics the inter-subunit oligomerization motif of RAD51 [10]. Through this mode of interaction, the BRC is antagonistic to RAD51 nucleoprotein filament formation [12]. But, biochemical analyses have revealed that the BRC can also stimulate RAD51 oligomerization on DNA [13]. Studies on RAD51-catalyzed strand pairing have shown that BRC elements can both stimulate RAD51 filament formation on ssDNA, yet also inhibit assembly on dsDNA, to promote the sequential progression of the DNA strand exchange reaction [13]. BRC repeats do not all act in an equivalent manner. It was found that the eight isolated individual BRC repeats from human BRCA2 fall into two classes [14]. Members of one class comprising BRC1, -2, -3, and -4 bind tightly to free RAD51, while members of the second group comprising BRC5, -6, -7, and -8 bind poorly to free RAD51 but tightly with RAD51 nucleoprotein filaments on ssDNA. Each member of the first class inhibits the RAD51 ATPase and thus can stabilize the extended form of the RAD51-ssDNA filament that is active in strand exchange. These aforementioned biochemical studies were performed using short BRC peptides fused to GST-tags to enable purification and pulldown methodology for in vitro experimentation into RAD51 complex formation and interplay with DNA. Other approaches to understanding more about structural determinants important in BRC-RAD51 interaction have come from experimental-based assays [15], [16] as well as computational methods coupled with various biochemical and biophysical analytical procedures often in conjunction with short synthetic BRC peptides to test predictions [11], [17], [18], [19], [20], [21].

Despite great strides made towards elucidating BRC function, many facets of BRC elements remain poorly understood. For instance, it is not clear why the number of BRC repeats is variable from one organism to the next. Human BRCA2, as mentioned has eight highly related, non-identical repeats that fall into two functional classes, but what the sequence determinants are for a particular BRC to fall into one class or the other is not clear. Even BRC elements of the same class do not appear to be functionally redundant, as a point mutation in a single BRC is enough to cause loss of BRCA2 functional activity in human [6] (BRCA2 missense mutations can be viewed in the Catalogue of Somatic Mutations in Cancer-- https://cancer.sanger.ac.uk/cosmic). Yet, artificial BRCA2 constructs created by coupling a few or even a single BRC element to the heterologous DNA binding domain of RPA70 are capable of restoring a substantial level of BRCA2 DNA repair proficiency and suppressing cellular defects associated with BRCA2 mutant mammalian cells [9]. In a different twist, trypanosome BRCA2 has 15 BRC repeats, 14 of which are identical, but mutant cells expressing a variant deleted of all but four BRCs function as efficiently as the full-length protein in DNA repair [22]. However, a variant with only a single BRC fails to complement. But, in yet another twist, the BRCA2 family member Brh2 of the fungus Ustilago maydis has only a single BRC element, which is necessary and sufficient for function [23].

The variation of BRC repeat number and sequence in BRCA2 family members throughout the eukaryotic domain has been a source of fascination for us as investigators of Brh2 and the DNA repair and homologous recombination system in U. maydis. Prior studies from this laboratory have documented the remarkable plasticity of Brh2 in being able to function in the absence of the native DBD or with an alternative heterologous DNA binding domain [8]. But, aside from establishing that the BRC element is important for DNA repair, detailed analysis of its function has not been undertaken. Structural and computational studies with the isolated human BRC4 and other repeats make predictions about the functional contribution of particular residues. Here, as an approach to understanding BRC function, we were interested to test if the rules learned from the HsBRC4 model system might extend to the single BRC element of Brh2. Our strategy was to use those findings to guide us in determining the contribution of individual residues to BRC function in the context of the full-length Brh2 protein by measuring DNA repair proficiency of a brh2Δ mutant expressing BRC variants with different amino acid substitutions. We report here a number of features of the BRC that mark the importance of particular residues in BRC function and that allow us to rank their contribution directly to DNA repair proficiency as governed by Brh2.