Ashok R. Venkitaraman

Cancer Susceptibility and the Functions of BRCA1 and BRCA2

Inherited mutations in BRCA1 or BRCA2 predispose to breast, ovarian, and other cancers. Their ubiquitously expressed protein products are implicated in processes fundamental to all cells, including DNA repair and recombination, checkpoint control of cell cycle, and transcription. Here, I examine what is known about the biological functions of the BRCA proteins and ask how their disruption can induce susceptibility to specific types of cancer.

AURORA-A amplification overrides the mitotic spindle assembly checkpoint, inducing resistance to Taxol

The serine-threonine kinase gene AURORA-A is commonly amplified in epithelial malignancies. Here we show that elevated Aurora-A expression at levels that reflect cancer-associated gene amplification overrides the checkpoint mechanism that monitors mitotic spindle assembly, inducing resistance to the chemotherapeutic agent paclitaxel (Taxol). Cells overexpressing Aurora-A inappropriately enter anaphase despite defective spindle formation, and the persistence of Mad2 at the kinetochores, marking continued activation of the spindle assembly checkpoint. Mitosis is subsequently arrested by failure to complete cytokinesis, resulting in multinucleation. This abnormality is relieved by an inhibitory mutant of BUB1, linking the mitotic abnormalities provoked by Aurora-A overexpression to spindle checkpoint activity. Consistent with this conclusion, elevated Aurora-A expression causes resistance to apoptosis induced by Taxol in a human cancer cell line.

Involvement of Brca2 in DNA Repair

Abnormalities precipitated by a targeted truncation in the murine gene Brca2 define its involvement in DNA repair. In culture, cells harboring truncated Brca2 exhibit a proliferative impediment that worsens with successive passages. Arrest in the G1 and G2/M phases is accompanied by elevated p53 and p21 expression. Increased sensitivity to genotoxic agents, particularly ultraviolet light and methylmethanesulfonate, shows that Brca2 function is essential for the ability to survive DNA damage. But checkpoint activation and apoptotic mechanisms are largely unaffected, thereby implicating Brca2 in repair. This is substantiated by the spontaneous accumulation of chromosomal abnormalities, including breaks and aberrant chromatid exchanges. These findings define a function of Brca2 in DNA repair, whose loss precipitates replicative failure, mutagen sensitivity, and genetic instability reminiscent of Bloom syndrome and Fanconi anemia.

Role of BRCA2 in Control of the RAD51 Recombination and DNA Repair Protein

Individuals carrying BRCA2 mutations are predisposed to breast and ovarian cancers. Here, we show that BRCA2 plays a dual role in regulating the actions of RAD51, a protein essential for homologous recombination and DNA repair. First, interactions between RAD51 and the BRC3 or BRC4 regions of BRCA2 block nucleoprotein filament formation by RAD51. Alterations to the BRC3 region that mimic cancer-associated BRCA2 mutations fail to exhibit this effect. Second, transport of RAD51 to the nucleus is defective in cells carrying a cancer-associated BRCA2 truncation. Thus, BRCA2 regulates both the intracellular localization and DNA binding ability of RAD51. Loss of these controls following BRCA2 inactivation may be a key event leading to genomic instability and tumorigenesis.

Insights into DNA recombination from the structure of a RAD51-BRCA2 complex

The breast cancer susceptibility protein BRCA2 controls the function of RAD51, a recombinase enzyme, in pathways for DNA repair by homologous recombination. We report here the structure of a complex between an evolutionarily conserved sequence in BRCA2 (the BRC repeat) and the RecA-homology domain of RAD51. The BRC repeat mimics a motif in RAD51 that serves as an interface for oligomerization between individual RAD51 monomers, thus enabling BRCA2 to control the assembly of the RAD51 nucleoprotein filament, which is essential for strand-pairing reactions during DNA recombination. The RAD51 oligomerization motif is highly conserved among RecA-like recombinases, highlighting a common evolutionary origin for the mechanism of nucleoprotein filament formation, mirrored in the BRC repeat. Cancer-associated mutations that affect the BRC repeat disrupt its predicted interaction with RAD51, yielding structural insight into mechanisms for cancer susceptibility.

EMSY Links the BRCA2 Pathway to Sporadic Breast and Ovarian Cancer

The BRCA2 gene is mutated in familial breast and ovarian cancer, and its product is implicated in DNA repair and transcriptional regulation. Here we identify a protein, EMSY, which binds BRCA2 within a region (exon 3) deleted in cancer. EMSY is capable of silencing the activation potential of BRCA2 exon 3, associates with chromatin regulators HP1beta and BS69, and localizes to sites of repair following DNA damage. EMSY maps to chromosome 11q13.5, a region known to be involved in breast and ovarian cancer. We show that the EMSY gene is amplified almost exclusively in sporadic breast cancer (13%) and higher-grade ovarian cancer (17%). In addition, EMSY amplification is associated with worse survival, particularly in node-negative breast cancer, suggesting that it may be of prognostic value. The remarkable clinical overlap between sporadic EMSY amplification and familial BRCA2 deletion implicates a BRCA2 pathway in sporadic breast and ovarian cancer.

HP1-β mobilization promotes chromatin changes that initiate the DNA damage response

Minutes after DNA damage, the variant histone H2AX is phosphorylated by protein kinases of the phosphoinositide kinase family, including ATM, ATR or DNA-PK. Phosphorylated (γ)-H2AX—which recruits molecules that sense or signal the presence of DNA breaks, activating the response that leads to repair—is the earliest known marker of chromosomal DNA breakage. Here we identify a dynamic change in chromatin that promotes H2AX phosphorylation in mammalian cells. DNA breaks swiftly mobilize heterochromatin protein 1 (HP1)-β (also called CBX1), a chromatin factor bound to histone H3 methylated on lysine 9 (H3K9me). Local changes in histone-tail modifications are not apparent. Instead, phosphorylation of HP1-β on amino acid Thr 51 accompanies mobilization, releasing HP1-β from chromatin by disrupting hydrogen bonds that fold its chromodomain around H3K9me. Inhibition of casein kinase 2 (CK2), an enzyme implicated in DNA damage sensing and repair, suppresses Thr 51 phosphorylation and HP1-β mobilization in living cells. CK2 inhibition, or a constitutively chromatin-bound HP1-β mutant, diminishes H2AX phosphorylation. Our findings reveal an unrecognized signalling cascade that helps to initiate the DNA damage response, altering chromatin by modifying a histone-code mediator protein, HP1, but not the code itself.

Impaired immunoglobulin gene rearrangement in mice lacking the IL-7 receptor

To generate the full diversity of antibody heavy-chain genes, hundreds of dispersed germline V segments must undergo recombination following D–J segment joining. Here we report that this process is regulated by the α-chain of the receptor for interleukin-7, a cytokine that stimulates B-cell lymphopoiesis. D–J joining occurs normally in immature B lymphocytes from mice lacking the α-chain of the interleukin-7 receptor (IL-7Rα). But recombination of V segments is progressively impaired as their distance increases upstream of D/J, causing infrequent rearrangement of most V segments, which markedly reduces diversity. This is not simply due to defective cell proliferation or impaired recombinase expression. Rather, germline transcripts from distal, unrearranged V segments, a marker of chromatin changes that precede recombination, are specifically silenced. So too is expression of Pax-5, which binds to heavy-chain locus control elements and normally stimulates recombination, suggesting a mechanism for these effects. Thus ligands of the interleukin-7 receptor deliver an extrinsic signal that targets V segment recombination in the heavy-chain locus by altering the accessibility of DNA substrates to the recombinase. This mechanism augments the recombinational diversity of the primary antibody repertoire.

Full-length archaeal Rad51 structure and mutants: Mechanisms for RAD51 assembly and control by BRCA2

To clarify RAD51 interactions controlling homologous recombination, we report here the crystal structure of the full‐length RAD51 homolog from Pyrococcus furiosus. The structure reveals how RAD51 proteins assemble into inactive heptameric rings and active DNA‐bound filaments matching three‐dimensional electron microscopy reconstructions. A polymerization motif (RAD51‐PM) tethers individual subunits together to form assemblies. Subunit interactions support an allosteric ‘switch’ promoting ATPase activity and DNA binding roles for the N‐terminal domain helix–hairpin–helix (HhH) motif. Structural and mutational results characterize RAD51 interactions with the breast cancer susceptibility protein BRCA2 in higher eukaryotes. A designed P.furiosus RAD51 mutant binds BRC repeats and forms BRCA2‐dependent nuclear foci in human cells in response to γ‐irradiation‐induced DNA damage, similar to human RAD51. These results show that BRCA2 repeats mimic the RAD51‐PM and imply analogous RAD51 interactions with RAD52 and RAD54. Both BRCA2 and RAD54 may act as antagonists and chaperones for RAD51 filament assembly by coupling RAD51 interface exchanges with DNA binding. Together, these structural and mutational results support an interface exchange hypothesis for coordinated protein interactions in homologous recombination.

Mitotic Checkpoint Inactivation Fosters Transformation in Cells Lacking the Breast Cancer Susceptibility Gene, Brca2

The murine Brca2 gene encodes a nuclear protein implicated in DNA repair. Brca2 behaves as a tumor suppressor, but paradoxically, its truncation causes proliferative arrest and spontaneous chromosomal damage. Here, we report that inactivation of cell cycle checkpoints responsive to mitotic spindle disruption, by mutant forms of p53 or Bub1, relieves growth arrest and initiates neoplastic transformation in primary cells homozygous for truncated Brca2. Tumors from Brca2-deficient animals exhibit dysfunction of the spindle assembly checkpoint, accompanied by mutations in p53, Bub1, and Mad3L. The chromosomal aberrations precipitated by Brca2 truncation can be suppressed by mutant forms of Bub1 and p53. Thus, inactivating mutations in mitotic checkpoint genes likely cooperate with BRCA2 deficiency in the pathogenesis of inherited breast cancer, with important implications for treatment.