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

Abstract

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.

Mutation of a BRCT domain selectively disrupts DNA single-strand break repair in noncycling Chinese hamster ovary cells

“…3C), suggesting that the BRCT II domain fulfilled an additional role in SSBR. To examine this possibility further, we compared the effect on SSBR of the two mutations that comprise XRCC1 pmBRCT because, although one of these ablates binding to Lig-3 (VI584͞585DD), the other (W611D) does not (6). We also examined the effect on SSBR of a deletion mutation (⌬529-633) that completely removes the BRCT II domain (5).…”

“…The mutation W611D removes the tryptophan residue that is largely invariant among BRCT domains and is believed to disrupt proper folding of XRCC1 BRCT II (12). This mutation does not ablate interaction with Lig-3, however (6). Finally, we also examined the effect on SSBR of a deletion mutation that removes the entire BRCT II domain (5).…”

“…This structure binds Lig-3 and in so doing maintains normal levels of the DNA ligase (4-7). In cells lacking XRCC1 or expressing XRCC1 that cannot interact with Lig-3, the latter polypeptide is degraded, resulting in 4 to 6-fold reduced levels of the DNA ligase (4,6,13). Thus, one role served by the BRCT II domain is to maintain high cellular levels of Lig-3.…”

“…XRCC1 protein interacts with DNA ligase III (Lig-3) and maintains normal cellular levels of this polypeptide (3,4). The interaction between XRCC1 and Lig-3 is mediated via BRCT domains located at the C terminus of both polypeptides (5,6). Intriguingly, there is a cell-cycle stage-specific requirement in mammalian cells for the C-terminal XRCC1 BRCT domain (denoted BRCT II), as it is required for XRCC1-dependent SSBR during G 1 but is dispensable for this process in S phase (7).…”

Tyrosyl-DNA Phosphodiesterase as a Target for Anticancer Therapy

“…In addition, the interaction of XRCC1 with PNKP has been shown to stimulate both the 5′-kinase and 3′-phosphatase activities of this enzyme [57]. Similarly, XRCC1 interacts with DNA ligase III and increases the intracellular stability of the ligase [58,59]. The association PARP-1 with the XRCC1 complex has also been observed.…”