These results demonstrate that expression of truncated Bard1 peptides which disrupt the endogenous Brca1-Bard1 heterodimer can significantly decrease HDR of a chromosome DSB. That this effect is specific to the Brca1-Bard1 heterodimer is supported by results with a mutated peptide hB202-L107P, which is disrupted at the interface for interaction with BRCA1. As predicted, the hB202-L107P mutation completely disrupted the ability to interact with BRCA1 and did not affect repair by gene conversion of an induced DSB, despite being highly expressed and localized to the nucleus. From the HDR results, along with the MMC hypersensitivity of cells that stably express the truncated Bard1 peptide, we conclude that BARD1 participates with BRCA1 in the HDR of an induced chromosome break through its RING-RING interaction with BRCA1. Thus, these results implicate Bard1 sequences downstream from the RING domain for proper HDR by the complex.
The methods used to assess repair in mammalian cells typically involve the delivery of genotoxic damage through radiation or drugs, followed by cell survival assays or immunocytochemical detection of protein relocalization. However, these assays do not provide the molecular details required to assess specific repair pathways. Many DNA damage response proteins colocalize with BRCA1 to sites of damage (51
). The function of many of these colocalizing proteins includes protein activation and recruitment to the damage site, whereas others are involved in repair of the damage. The proteins involved in HDR, as demonstrated by repair assays that can assess the specific repair defects, include Rad51, BRCA1, BRCA2 (32
), and now BARD1.
Truncated BARD1 peptides similar in size to our dominant-negative peptides have been shown to augment the E3 ubiquitin ligase activity of BRCA1 (8
). Thus, although truncated BARD1 peptides appear to be sufficient for the intrinsic enzymatic function of the BRCA1-BARD1 heterodimer, the HDR function of the heterodimer appears to require full-length BARD1. The E3 ubiquitin ligase activity may therefore be separable from the HDR function of the heterodimer or, conversely, specific enzymatic substrates may require the full-length BRCA1-BARD1 heterodimer. Few in vivo substrates have yet been defined that undergo BRCA1-BARD1-mediated ubiquitination; however, autoubiquitination of the heterodimer or another protein associated with the complex has been noted (8
). More importantly, the downstream effects of proteins that are ubiquitinated by the BRCA1-BARD1 heterodimer remain undefined.
mutant ES cell line we examined, like other BRCA1 mutant cells, is hypersensitive to cisplatin and MMC and incurs spontaneous chromosome instability (3
). As noted above, the Brca1−/−
ES cells, as well as murine embryonic fibroblasts with a similar exon 11-deleted isoform, have diminished but detectable damage-induced Rad51 foci (3
). Because expression of the truncated Bard1 peptides in the Brca1-deficient cells further diminished the repair of induced DSBs by HDR, the Brca1Δ11
peptide appears to be a hypomorph for HDR function, implying that Brca1-null cells, if viable, would be severely reduced for HDR. Interestingly, the Brca1-deficient murine embryonic fibroblasts did not appear to undergo phosphorylation of the Brca1Δ11
peptide after DNA damage (16
), indicating that damage-induced phosphorylation may not be necessary for the residual HDR activity.
These results have several implications. In the Brca1-conditional mammary tumor model, in which a proportion of the mammary epithelial cells express a Brca1Δ11
product, mammary tumors arose at an incidence of 25% (4
). Given the hypomorphic HDR function of the Brca1Δ11
product, a greater reduction in cellular repair capability may be expected to have more profound effects, by increasing the mammary tumor incidence in the Brca1Δ11
-conditional mouse model or, alternatively, leading to a rapid and overwhelming accumulation of chromosome damage and cell death, a result more akin to the phenotype observed in Brca1-deficient embryonic tissue (9
). Notably, RAD51, the central protein of HDR, has not been found to be mutated or lost in human tumors (39
). These results also raise the issue of whether the endogenously occurring BRCA1 spliced isoforms in humans that retain the RING and BRCT domains act to preserve genetic stability through residual repair function or are tumor promoting in the absence of full-length BRCA1 by allowing the propagation of chromosomally damaged cells. Since the variation in cancer incidence observed in clinical populations may be related to specific mutations that harbor residual function, it is important to further decipher these structure-function relationships.
Tumor-associated missense mutations in BRCA1 include cysteine residues involved in zinc binding, although thus far the zinc-binding residues in BARD1 have not been found to be mutated in human tumors (13
). Although the tumor associated C61G mutation in BRCA1 abolishes the ubiquitin ligase activity of the BRCA1-BARD1 heterodimeric RING (15
), the corresponding mutation in BARD1, C83G, results in a slight reduction or no reduction in ubiquitin ligase activity (15
). In addition, the corresponding Xenopus
BARD1 C77G mutation did not abolish the xBRCA1-xBARD1 interaction in frog embryos (22
). The intermediate reduction in gene conversion repair we observed with the hB202-C83G peptide and the lack of a repair defect with full-length hBARD1-C83G could potentially be explained by decreased protein expression observed by Western blot analysis. As the hB202 peptide itself reduces gene conversion repair, the reduced level of gene conversion with the hB202-C83G peptide may reflect the dominant-negative effect of the truncation rather than an effect due to the point mutation. The reason for the observed lower expression levels of this mutant protein in both the full-length and truncated BARD1 constructs is not evident, especially in light of the strong interaction with the BRCA1 peptide observed in the mammalian two-hybrid experiments. However, a mutation in the corresponding cysteine residue in BRCA1 is substantially more sensitive to proteolytic degradation in vitro, presumably due to compromised zinc binding (5
); thus, it is possible that the BARD1 C83G mutation could similarly affect protein stability in vivo.
A search for BARD1 mutations in both familial and sporadic cancers has yielded few data to support BARD1 as a commonly mutated tumor suppressor in human tumors (13
). A germ line missense mutation occurring in BARD1 was found in a patient with late-onset cancer of the ovary, breast, and endometrial tissues, with loss of the wild-type allele confirmed in the ovarian tumor (52
). This mutation, Q564H, is located between the ankyrin and the BRCT repeats at a residue that is conserved between mouse and human BARD1. Functional analysis has shown that this mutation does not interfere with binding of BARD1 to BRCA1; however, it exhibits decreased binding to CstF-50 and disrupts the ability of BARD1 to inhibit 3′-cleavage of mRNA precursors (24
). In our assays, overexpression of BARD1-Q564H did not diminish gene conversion repair of an induced DSB; therefore, this mutant BARD1 does not exhibit dominant-negative activity in the presence of wild-type BARD1. This result does not rule out the possibility that the BARD1 Q564H mutation could have an effect on HDR in the absence of wild-type BARD1 or a tumor-promoting effect through impaired regulation of mRNA processing. However, the mutation appears to be a rare event, and its role in tumorigenesis needs confirmation. Since Bard1-deficient cells have not been identified and the Bard1 knockout results in early embryonic lethality (28
), hypomorphic alleles that allow for cellular viability may be necessary to further elucidate the effect of specific BARD1 mutations.
It is hypothesized that defects in HDR lead to unrepaired and misrepaired chromosomes during the normal course of DNA replication, thereby leading toward increased chromosome instability and an increased propensity for tumorigenesis. This spontaneous chromosome instability has been observed in cells deficient for BRCA1 and BRCA2 and mouse models that are either hypomorphic or have tissue-restricted deletions of either gene develop cancer (19
). However, chromosome instability is also observed in cells and mouse models with a spectrum of deficiencies, many of which involve the cellular response to DNA damage (11
). The importance of identifying at the molecular level how this instability arises and how each protein functions within the DNA damage response pathway that leads to genomic instability becomes increasingly important since not all of these deficiencies have been documented to predispose to tumorigenesis.