Role of a BRCT domain in the interaction of DNA ligase III-alpha with the DNA repair protein XRCC1

The BRCT domain (for BRCA1 carboxyl terminus) is a protein motif of unknown function, comprising approximately 100 amino acids in five conserved blocks denoted A-E. BRCT domains are present in the tumour suppressor protein BRCA1 [1-3], and the domain is found in over 40 other proteins, defining a superfamily that includes DNA ligase III-alpha and the essential human DNA repair protein XRCC1. DNA ligase III-alpha and XRCC1 interact via their carboxyl termini, close to or within regions that contain a BRCT domain [4]. To examine whether the primary role of the carboxy-terminal BRCT domain of XRCC1 (denoted BRCT II) is to mediate the interaction with DNA ligase III-alpha, we identified the regions of the domain that are required and sufficient for the interaction. An XRCC1 protein in which the conserved D-block tryptophan was disrupted by point mutation retained the ability to interact with DNA ligase III-alpha, so this tryptophan must mediate a different, although conserved, role. XRCC1 in which the weakly conserved C-block was mutated lost the ability to interact with DNA ligase III-alpha. Moreover, 20 amino acids spanning the C-block of BRCT II conferred full DNA ligase III-alpha binding activity upon an unrelated polypeptide. An XRCC1 protein in which this 20mer was deleted could not maintain normal levels of DNA ligase III-alpha in transfected rodent cells, a phenotype associated with defective repair [5]. In summary, these data demonstrate that a BRCT domain can mediate a biologically important protein-protein interaction, and support the existence of additional roles.     

Identification of a BRCA1-associated kinase with potential biological relevance

A biochemical approach was used to identify proteins which interact with human BRCA1. Through this work, a kinase activity which co-purifies with BRCA1 has been identified. This kinase activity, which phosphorylates BRCA1 in vitro, was originally identified in Sf9 insect cells but is also present in cells of human origin including breast and ovarian carcinoma cell lines. The BRCA1 kinase activity in vitro is associated with a fragment of BRCA1 encompassing amino acids 329-435. This peptide is also phosphorylated in various human cell lines. A computer-assisted sequence analysis revealed that this peptide was a potential substrate for phosphorylation by PKA, PKC, or CKII. However, phosphorylation by these kinases could not be demonstrated in vitro indicating the presence of another kinase activity. Phosphorylation in vitro requires a minimal domain of BRCA1 encompassing amino acids 379-408. Notably, deletion of this minimal domain abolishes growth suppression by BRCA1 indicating that this domain, as well as phosphorylation within this domain, may be important for BRCA1 function.