Impaired DNA Damage Response in Cells Expressing an Exon 11-Deleted Murine Brca1 Variant That Localizes to Nuclear Foci

Abstract

Both human and mouse cells express an alternatively spliced variant of BRCA1, BRCA1-⌬11, which lacks exon 11 in its entirety, including putative nuclear localization signals. Consistent with this, BRCA1-⌬11 has been reported to reside in the cytoplasm, a localization that would ostensibly preclude it from playing a role in the nuclear processes in which its full-length counterpart has been implicated. Nevertheless, the finding that murine embryos bearing homozygous deletions of exon 11 survive longer than embryos that are homozygous for Brca1 null alleles suggests that exon 11-deleted isoforms may perform at least some of the functions of Brca1. We have analyzed both the full-length and the exon 11-deleted isoforms of the murine Brca1 protein.Our results demonstrate that full-length murine Brca1 is identical to human BRCA1 with respect to its cell cycle regulation, DNA damage-induced phosphorylation, nuclear localization, and association with Rad51. Surprisingly, we show that endogenous Brca1-⌬11 localizes to discrete nuclear foci indistinguishable from those found in wild-type cells, despite the fact that Brca1-⌬11 lacks previously defined nuclear localization signals. However, we further show that DNA damage-induced phosphorylation of Brca1-⌬11 is significantly reduced compared to full-length Brca1, and that gamma irradiation-induced Rad51 focus formation is impaired in cells in which only Brca1-⌬11 is expressed. Our results suggest that the increased viability of embryos bearing homozygous deletions of exon 11 may be due to expression of Brca1-⌬11 and suggest an explanation for the genomic instability that accompanies the loss of full-length Brca1.Germ line mutations in BRCA1 predispose women to earlyonset breast and ovarian cancers (18,38). The BRCA1 gene is composed of 23 exons that encode a 1,863-amino-acid fulllength protein, over half of which is encoded by an unusually large exon, exon 11, which is 3.4 kb in length. In addition to the full-length BRCA1 protein, p220 BRCA1 , human cells contain alternatively spliced variants referred to as BRCA1-⌬11 (referred to here as p97 BRCA1 ) and BRCA1-⌬11b (referred to here as p110 BRCA1 ), which lack all and most of exon 11, respectively (54, 58). These isoforms arise from in-frame splicing events and retain the highly conserved amino-terminal RING finger and carboxyl-terminal BRCT domains found in fulllength BRCA1 but lack the nuclear localization signals previously identified in exon 11 (11,54,58). The abundant expression of p97 BRCA1 and p110 BRCA1 has been demonstrated in a variety of adult tissues, including the human mammary gland, in which transcripts encoding p110 BRCA1 are expressed at levels comparable to those encoding p220 BRCA1 (33,54,58). The observation that human BRCA1 is phosphorylated in response to UV light, ionizing radiation, and other agents that damage DNA, and the identification of BRCA1-interacting proteins such as RAD51 and RAD50-Mre11-p95 complexes that colocalize with BRCA1 following DNA damage have suggested a role for BRCA1 in DNA r...

BRCA1 gene in breast cancer

“…Consistent with these findings, mouse embryonic fibroblasts (MEFs) carrying a targeted homozygous deletion of exon 11 (which contains ffi 60% of the Brca1 coding sequence) exhibited a defective G2/M checkpoint and extensive chromosomal anomalies (Huber et al, 2001). These Brca1-defective MEFs showed centrosome amplification with multiple functional centrosomes, which led to unequal chromosome segregation and aneuploidy (Xu et al, 1999b).…”

“…Fan et al (2001b) found that a truncated BRCA1 protein containing only the N-terminus (aa 1-302) enters the nucleus. Similarly, a BRCA1 mutant protein corresponding to a naturally occuring isoform (Brca1 D exon 11) enters the nucleus despite the fact that this isoform is missing both NLS1 and NLS2 (Huber et al, 2001). These findings suggest the presence of a cryptic (i.e., non-classical) NLS within the first 300 or so aa of BRCA1.…”

“…The best characterized of these is the 4.6 kb alternative splice product missing nucleotides 672-4,092, which encodes a truncated protein of about 97 kDa. The BRCA1 D exon 11 isoform is missing two classical nuclear localization signals (NLS), but recent studies suggest this isoform can still localize to the nucleus and retains some BRCA1 functional activity (Huber et al, 2001). …”

“…The latent period for BRCA1 GENE IN BREAST CANCER mammary tumorigenesis was significantly decreased in mice heterozygous for p53 (p53 þ/À ). Subsequent studies revealed that a truncated (92 kDa) Brca1 DEx11/DEx11 protein was expressed in the Brca1 mutant mouse embryo fibroblasts (MEFs) and that this protein localized to the nucleus (Huber et al, 2001). The Brca1 mutant MEFs proliferated normally in culture but displayed a defect in the Brca1-mediated DNA damage response.…”

BRCA1, PARP, and 53BP1: conditional synthetic lethality and synthetic viability

“…The Brca1 2D11 mutation has an in-frame deletion of exon 11 that results in production of a hypomorphic protein resulting from in-frame splicing from exon 10 to exon 12. This BRCA1 D11 variant has an intact RING domain, tandem BRCT-repeats and localizes to the nucleus, but is associated with a defect in HR-mediated repair (Huber et al, 2001). Unlike homozygous null-BRCA1 mutants, which show early embryonic lethality that is only modestly delayed by being placed in a p53 2/2 background, homozygous Brca1 D11/D11 mutants show mid-gestational embryonic lethality that can be reversed in certain strain backgrounds or by breeding to a p53 +/2 genotype (Cressman et al, 1999a;Xu et al, 2001).…”