BRCA1 C-terminal Research Articles

The DNA Damage Response Mediator MDC1 Directly Interacts with the Anaphase-promoting Complex/Cyclosome

MDC1 (NFBD1), a mediator of the cellular response to DNA damage, plays an important role in checkpoint activation and DNA repair. Here we identified a cross-talk between the DNA damage response and cell cycle regulation. We discovered that MDC1 binds the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that controls the cell cycle. The interaction is direct and is mediated by the tandem BRCA1 C-terminal domains of MDC1 and the C terminus of the Cdc27 (APC3) subunit of the APC/C. It requires the phosphorylation of Cdc27 and is enhanced after induction of DNA damage. We show that the tandem BRCA1 C-terminal domains of MDC1, known to directly bind the phosphorylated form of histone H2AX (gamma-H2AX), also bind the APC/C by the same mechanism, as phosphopeptides that correspond to the C termini of gamma-H2AX and Cdc27 competed with each other for the binding to MDC1. Our results reveal a link between the cellular response to DNA damage and cell cycle regulation, suggesting that MDC1, known to have a role in checkpoint regulation, executes part of this role by binding the APC/C.

Study on the Molecular Structure and Bio-Activity (DNA Polymerase Inhibitory Activity, Anti-Inflammatory Activity and Anti-Oxidant Activity) Relationship of Curcumin Derivatives

We found that phenolic compounds, petasiphenol and curcumin (diferuloylmethane), were selective inhibitors of DNA polymerase λ (pol λ) in vitro. In this review, we described the molecular structure and bio-activity (i.e., pol inhibitory activity, antiinflammatory activity and anti-oxidant activity) relationship of phenolic compounds such as petasiphenol, curcumin and 13 chemically synthesized derivatives of curcumin. The inhibitory effect on pol λ (full-length, i.e. intact pol λ including the BRCA1 C-terminus (BRCT) domain) by some derivatives was stronger than that by curcumin, and monoacetylcurcumin (compound (13)) was the strongest pol λ inhibitor of all the compounds tested, achieving 50% inhibition at a concentration of 3.9 μM. Compound (13) did not inhibit the activity of the C-terminal catalytic domain of pol λ including the pol β-like core, in which the proline-rich region and BRCT domain were deleted from its N-terminal region. These curcumin derivatives had anti-12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammatory activity with the same tendency as pol λ inhibitory activity, but compound (13) had no anti-oxidant activity (i.e., DPPH radical-scavenging activity). This compound did not influence the activities of replicative pols such as α, δ and η, and had no effect on repair-related pol activity such as pol β, although the three-dimensional structure of pol β is thought to be highly similar to that of pol λ. Based on these results, the pol λ-inhibitory mechanism of compound (13) and the structure-activity relationship of anti-inflammation are discussed. Keywords: Curcumin, Monoacetylcurcumin, DNA polymerase λ, DNA polymerase inhibitory activity, Anti-inflammatory activity, Antioxidant activity, Enzyme inhibitor, TPA (12-O-tetradecanoylphorbol-13-acetate)

The role of CDK in the initiation step of DNA replication in eukaryotes

Cyclin-dependent kinases (CDKs) regulate the progression of the cell cycle in eukaryotes. One of the major roles of CDK is to promote chromosomal DNA replication. However, how CDKs promote DNA replication has been a long-standing question, because all the essential CDK substrates in DNA replication have not been identified yet. Recently Sld2 and Sld3 were identified as essential substrates of CDKs in the initiation step of DNA replication in budding yeast. Moreover, bypass of their phosphorylations is sufficient to promote DNA replication. Phosphorylation of Sld2 and Sld3 by CDKs enhances the formation of complex(es) with a BRCT (BRCA1 C-Terminal)-containing replication protein, Dpb11. We further propose that multiple phosphorylation by CDKs controls this process in budding yeast. Even though Sld3 orthologues in multicellular eukaryotes have not been identified, similar complex formation and, therefore, a similar mechanism of initiation control might be employed in eukaryotes.

The DNA topoisomerase IIβ binding protein 1 (TopBP1) interacts with poly (ADP‐ribose) polymerase (PARP‐1)

We investigated the physical association of the DNA topoisomerase IIbeta binding protein 1 (TopBP1), involved in DNA replication and repair but also in regulation of apoptosis, with poly(ADP-ribose) polymerase-1 (PARP-1). This enzyme plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. It was shown that the sixth BRCA1 C-terminal (BRCT) domain of TopBP1 interacts with a protein fragment of PARP-1 in vitro containing the DNA-binding and the automodification domains. More significantly, the in vivo interaction of endogenous TopBP1 and PARP-1 proteins could be shown in HeLa-S3 cells by co-immunoprecipitation. TopBP1 and PARP-1 are localized within overlapping regions in the nucleus of HeLa-S3 cells as shown by immunofluorescence. Exposure to UVB light slightly enhanced the interaction between both proteins. Furthermore, TopBP1 was detected in nuclear regions where poly(ADP-ribose) (PAR) synthesis takes place and is ADP-ribosylated by PARP-1. Finally, cellular (ADP-ribosyl)ating activity impairs binding of TopBP1 to Myc-interacting zinc finger protein-1 (Miz-1). The results indicate an influence of post-translational modifications of TopBP1 on its function during DNA repair.

Functional Impact of Missense Variants in BRCA1 Predicted by Supervised Learning

Many individuals tested for inherited cancer susceptibility at the BRCA1 gene locus are discovered to have variants of unknown clinical significance (UCVs). Most UCVs cause a single amino acid residue (missense) change in the BRCA1 protein. They can be biochemically assayed, but such evaluations are time-consuming and labor-intensive. Computational methods that classify and suggest explanations for UCV impact on protein function can complement functional tests. Here we describe a supervised learning approach to classification of BRCA1 UCVs. Using a novel combination of 16 predictive features, the algorithms were applied to retrospectively classify the impact of 36 BRCA1 C-terminal (BRCT) domain UCVs biochemically assayed to measure transactivation function and to blindly classify 54 documented UCVs. Majority vote of three supervised learning algorithms is in agreement with the assay for more than 94% of the UCVs. Two UCVs found deleterious by both the assay and the classifiers reveal a previously uncharacterized putative binding site. Clinicians may soon be able to use computational classifiers such as those described here to better inform patients. These classifiers can be adapted to other cancer susceptibility genes and systematically applied to prioritize the growing number of potential causative loci and variants found by large-scale disease association studies.

A mouse model of basal‐like breast carcinoma with metaplastic elements

Breast cancers arising in carriers of germline BRCA1 mutations frequently have a basal-like phenotype. Basal-like cancers are characterized by high histological grade, central necrotic areas, foci with metaplastic differentiation, lack of hormone receptor and HER2 (ErbB2) expression, and consistent positivity for basal markers, including CK5/6, CK14, and EGFR. We have used germline manipulation to generate a conditional mouse model of Brca1 deficiency. Transgenic expression of Cre recombinase in the mammary gland of these mice results in deletion of exons encoding the C-terminus of Brca1 and leads to tumour formation when combined with heterozygosity for a p53 mutation. Histologically, these mammary gland tumours were characterized by high histological grade, central necrotic areas, and presence of homologous metaplastic elements. These metaplastic elements consisted of neoplastic spindle cells or squamous cell differentiation in the form of keratin pearls or individual cell keratinization. Immunohistochemical analysis revealed expression of basal-like markers in all cases. The tumour phenotype generated in our mouse model was compared with published data on human basal-like breast carcinomas and also with metaplastic breast cancers with a basal-like phenotype; the comparison showed that we have generated a mouse model of basal-like breast cancer, which should prove useful in testing new and targeted treatments for this type of breast cancer.

“Similarity Trap” in Protein-Protein Interactions Could Be Carcinogenic: Simulations of p53 Core Domain Complexed with 53BP1 and BRCA1 BRCT Domains

Similar binding sites often imply similar protein-protein interactions and similar functions; however, similar binding sites may also constitute traps for nonfunctional associations. How are similar sites distinguished to prevent misassociations? BRCT domains from breast cancer-susceptibility gene product BRCA1 and protein 53BP1 have similar structures yet different binding behaviors with p53 core domain. 53BP1-BRCT domain forms a stable complex with p53. In contrast, BRCA1-p53 interaction is weak or other mechanisms operate. To delineate the difference, we designed 13 BRCA1-BRCT mutants and computationally investigated the structural and stability changes compared to the experimental p53-53BP1 structure. Interestingly, of the 13, the 2 mutations that are cancerous and involve nonconserved residues are those that enforced p53 core domain binding with BRCA1-BRCT in a way similar to p53-53BP1 binding. Hence, falling into the "similarity trap" may disrupt normal BRCA1 and p53 functions. Our results illustrate how this trap is avoided in the native state.

The tandem BRCT domain of 53BP1 is not required for its repair function.

53BP1 plays an important role in cellular response to DNA damage. It is thought to be the mammalian homologue of budding yeast Rad9 and/or fission yeast Crb2. Rad9/Crb2 are bona fide checkpoint proteins whose activation requires their corresponding C-terminal tandem BRCT (BRCA1 C-terminal) motifs, which mediate their oligomerization and phosphorylation at multiple sites following DNA damage. Here we show that the function of human 53BP1 similarly depends on its oligomerization and phosphorylation at multiple sites but in a BRCT domain-independent manner. Moreover, unlike its proposed yeast counterparts, human 53BP1 only has limited checkpoint functions but rather acts as an adaptor in the repair of DNA double strand breaks. This difference in function may reflect the higher complexity of the DNA damage response network in metazoa including the evolution of other BRCT domain-containing proteins that may have functions redundant or overlapping with those of 53BP1.

The critical mutagenic translesion DNA polymerase Rev1 is highly expressed during G 2 /M phase rather than S phase

The Rev1 protein lies at the root of mutagenesis in eukaryotes. Together with DNA polymerase zeta (Rev3/7), Rev1 function is required for the active introduction of the majority of mutations into the genomes of eukaryotes from yeast to humans. Rev1 and polymerase zeta are error-prone translesion DNA polymerases, but Rev1's DNA polymerase catalytic activity is not essential for mutagenesis. Rather, Rev1 is thought to contribute to mutagenesis principally by engaging in crucial protein-protein interactions that regulate the access of translesion DNA polymerases to the primer terminus. This inference is based on the requirement of the N-terminal BRCT (BRCA1 C-terminal) domain of Saccharomyces cerevisiae Rev1 for mutagenesis and the interaction of the C-terminal region of mammalian Rev1 with several other translesion DNA polymerases. Here, we report that S. cerevisiae Rev1 is subject to pronounced cell cycle control in which the levels of Rev1 protein are approximately 50-fold higher in G(2) and throughout mitosis than during G(1) and much of S phase. Differential survival of a rev1Delta strain after UV irradiation at various points in the cell cycle indicates that this unanticipated regulation is physiologically relevant. This unexpected finding has important implications for the regulation of mutagenesis and challenges current models of error-prone lesion bypass as a process involving polymerase switching that operates mainly during S phase to rescue stalled replication forks.

A Dbf4p BRCA1 C-Terminal-Like Domain Required for the Response to Replication Fork Arrest in Budding Yeast

Dbf4p is an essential regulatory subunit of the Cdc7p kinase required for the initiation of DNA replication. Cdc7p and Dbf4p orthologs have also been shown to function in the response to DNA damage. A previous Dbf4p multiple sequence alignment identified a conserved approximately 40-residue N-terminal region with similarity to the BRCA1 C-terminal (BRCT) motif called "motif N." BRCT motifs encode approximately 100-amino-acid domains involved in the DNA damage response. We have identified an expanded and conserved approximately 100-residue N-terminal region of Dbf4p that includes motif N but is capable of encoding a single BRCT-like domain. Dbf4p orthologs diverge from the BRCT motif at the C terminus but may encode a similar secondary structure in this region. We have therefore called this the BRCT and DBF4 similarity (BRDF) motif. The principal role of this Dbf4p motif was in the response to replication fork (RF) arrest; however, it was not required for cell cycle progression, activation of Cdc7p kinase activity, or interaction with the origin recognition complex (ORC) postulated to recruit Cdc7p-Dbf4p to origins. Rad53p likely directly phosphorylated Dbf4p in response to RF arrest and Dbf4p was required for Rad53p abundance. Rad53p and Dbf4p therefore cooperated to coordinate a robust cellular response to RF arrest.

β-Sitosterol-3- O-β- d-glucopyranoside: A eukaryotic DNA polymerase λ inhibitor

β-Sitosterol-3- O-β- d-glucopyranoside (compound 1), a steroidal glycoside isolated from onion ( Allium cepa L.) selectively inhibited the activity of mammalian DNA polymerase λ (pol λ) in vitro. The compound did not influence the activities of replicative DNA polymerases such as α, δ and ɛ, but also showed no effect even on the activity of pol β which is thought to have a very similar three-dimensional structure to the pol β-like region of pol λ. Since parts of compound 1 such as β-sitosterol (compound 2) and d-glucose (compound 3) did not influence the activities of any enzymes tested, the converted structure of compounds 2 and 3 might be important for pol λ inhibition. The inhibitory effect of compound 1 on both intact pol λ (i.e. residues 1–575) and a truncated pol λ lacking the N-terminal BRCA1 C-terminus (BRCT) domain (133–575, del-1 pol λ) was dose-dependent, and 50% inhibition was observed at a concentration of 9.1 and 5.4 μM, respectively. The compound 1-induced inhibition of del-1 pol λ activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol λ inhibitory mechanism of compound 1 is discussed.

Mapping and conformational characterization of the DNA-binding region of the breast cancer susceptibility protein BRCA1

The breast cancer susceptibility gene, BRCA1, encodes a large nuclear phosphoprotein, the major isoform of which is 1863 amino acids in size. Structure-function studies have been largely restricted to the only two domains identified by homology searches: the RING (really interesting new gene) and BRCT (BRCA1 C-terminus) domains. However, we have recently reported the identification of a large central soluble region of BRCA1 (residues 230-534) that binds specifically to four-way junction DNA, a property that potentially facilitates its role in the repair of DNA lesions by homologous recombination. We have now used a combination of limited proteolysis and extension cloning to identify more accurately the DNA-binding region of BRCA1. Limited trypsinolysis of BRCA1-(230-534) resulted in the production of a soluble domain identified as residues 230-339. However, after cloning, expression and purification of this region, studies revealed that it was unable to bind to four-way junctions, suggesting that the DNA-binding activity, in part, resides within residues 340-534. A series of fragments extending from residue 340 were produced, and each was tested for its ability to bind to four-way junction DNA in gel retardation assays. In these experiments, residues 340-554 of BRCA1 were identified as the minimal DNA-binding region. We then went on to characterize the conformation of this region using CD spectroscopy and analytical centrifugation.

5-(Hydroxymethyl)-2-furfural: A selective inhibitor of DNA polymerase λ and terminal deoxynucleotidyltransferase

5-(Hydroxymethyl)-2-furfural (HMF), a pyrolysate of carbohydrate isolated from instant coffee ( Coffea arabica L.), selectively inhibits the activities of mammalian DNA polymerase λ (pol λ) and terminal deoxynucleotidyltransferase (TdT) which are family X pols, in vitro. The compound influenced neither the activities of replicative DNA polymerases such as α, δ, and ε, nor even the activity of pol β which is from the same family and thought to have a very similar three-dimensional structure to the pol β-like region of pol λ. Since parts of HMF such as furan, furfuryl alcohol, and 2-furaldehyde did not influence the activities of any enzymes tested, the substituted form of furan with a hyroxymethyl group and a formyl group might be important for the inhibition of pol λ and TdT. The inhibitory effect of HMF on intact pol λ (i.e., residues 1–575), a truncated pol λ lacking the N-terminal BRCA1 C-terminus domain (133–575, del-1 pol λ) and another truncated pol λ lacking the N-terminal proline-rich region (245–575, del-2 pol λ) was dose-dependent, and 50% inhibition was observed at a concentration of 26.1, 10.3, and 4.6 μM, respectively. The IC 50 value of HMF for TdT was the same as that for del-2 pol λ (5.5 μM). The HMF-induced inhibition of both pol λ and TdT activities was competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, HMF was suggested to bind to the pol β-like region of pol λ and TdT.

Structural basis for phosphorylation-dependent signaling in the DNA-damage response

The response of eukaryotic cells to DNA damage requires a multitude of protein-protein interactions that mediate the ordered repair of the damage and the arrest of the cell cycle until repair is complete. Two conserved protein modules, BRCT and forkhead-associated (FHA) domains, play key roles in the DNA-damage response as recognition elements for nuclear Ser/Thr phosphorylation induced by DNA-damage-responsive kinases. BRCT domains, first identified at the C-terminus of BRCA1, often occur as multiple tandem repeats of individual BRCT modules. Our recent structural and functional work has revealed how BRCT repeats recognize phosphoserine protein targets. It has also revealed a secondary binding pocket at the interface between tandem repeats, which recognizes the amino-acid 3 residues C-terminal to the phosphoserine. We have also studied the molecular function of the FHA domain of the DNA repair enzyme, polynucleotide kinase (PNK). This domain interacts with threonine-phosphorylated XRCC1 and XRCC4, proteins responsible for the recruitment of PNK to sites of DNA-strand-break repair. Our studies have revealed a flexible mode of recognition that allows PNK to interact with numerous negatively charged substrates.

Inhibitory effect of tocotrienol on eukaryotic DNA polymerase λ and angiogenesis

Tocotrienols, vitamin E compounds that have an unsaturated side chain with three double bonds, selectively inhibited the activity of mammalian DNA polymerase λ (pol λ) in vitro. These compounds did not influence the activities of replicative pols such as α, δ, and ε, or even the activity of pol β which is thought to have a very similar three-dimensional structure to the pol β-like region of pol λ. Since δ-tocotrienol had the strongest inhibitory effect among the four (α- to δ-) tocotrienols, the isomer’s structure might be an important factor in the inhibition of pol λ. The inhibitory effect of δ-tocotrienol on both intact pol λ (residues 1–575) and a truncated pol λ lacking the N-terminal BRCA1 C-terminus (BRCT) domain (residues 133–575, del-1 pol λ) was dose-dependent, with 50% inhibition observed at a concentration of 18.4 and 90.1 μM, respectively. However, del-2 pol λ (residues 245–575) containing the C-terminal pol β-like region was unaffected. Tocotrienols also inhibited the proliferation of and formation of tubes by bovine aortic endothelial cells, with δ-tocotrienol having the greatest effect. These results indicated that tocotrienols targeted both pol λ and angiogenesis as anti-cancer agents. The relationship between the inhibition of pol λ and anti-angiogenesis by δ-tocotrienol was discussed.

Inhibition of Aurora-B kinase activity by poly(ADP-ribosyl)ation in response to DNA damage

The cell cycle-regulated Aurora-B kinase is a chromosomal passenger protein that is implicated in fundamental mitotic events, including chromosome alignment and segregation and spindle checkpoint function. Aurora-B phosphorylates serine 10 of histone H3, a function that has been associated with mitotic chromatin condensation. We find that activation of poly(ADP-ribose) polymerase (PARP) 1 by DNA damage results in a rapid block of H3 phosphorylation. PARP-1 is a NAD(+)-dependent enzyme that plays a multifunctional role in DNA damage detection and repair and maintenance of genomic stability. Here, we show that Aurora-B physically and specifically associates with the BRCT (BRCA-1 C-terminal) domain of PARP-1. Aurora-B becomes highly poly(ADP-ribosyl)ated in response to DNA damage, a modification that leads to a striking inhibition of its kinase activity. The highly similar Aurora-A kinase is not regulated by PARP-1. We propose that the specific inhibition of Aurora-B kinase activity by PARP-1 contributes to the physiological response to DNA damage.

Structural Basis for Cell Cycle Checkpoint Control by the BRCA1−CtIP Complex,

The breast and ovarian tumor suppressor BRCA1 has important functions in cell cycle checkpoint control and DNA repair. Two tandem BRCA1 C-terminal (BRCT) domains are essential for the tumor suppression activity of BRCA1 and interact in a phosphorylation-dependent manner with proteins involved in DNA damage-induced checkpoint control, including the DNA helicase BACH1 and the CtBP-interacting protein (CtIP). The crystal structure of the BRCA1 BRCT repeats bound to the PTRVSpSPVFGAT phosphopeptide corresponding to residues 322-333 of human CtIP was determined at 2.5 A resolution. The peptide binds to a cleft formed by the interface of the two BRCTs in a two-pronged manner, with phospho-Ser327 and Phe330 anchoring the peptide through extensive contacts with BRCA1 residues. Several hydrogen bonds and salt bridges that stabilize the BRCA1-BACH1 complex are missing in the BRCA1-CtIP interaction, offering a structural basis for the approximately 5-fold lower affinity of BRCA1 for CtIP compared to that of BACH1, as determined by isothermal titration calorimetry. Importantly, the side chain of Arg1775 in the cancer-associated BRCA1 mutation M1775R sterically clashes with the phenyl ring of CtIP Phe330, disrupting the BRCA1-CtIP interaction. These results provide new insights into the molecular mechanisms underlying the dynamic selection of target proteins involved in DNA repair and cell cycle control by BRCA1 and reveal how certain cancer-associated mutations affect these interactions.

Boundaries and physical characterization of a new domain shared between mammalian 53BP1 and yeast Rad9 checkpoint proteins

Eukaryotic cells have evolved DNA damage checkpoints in response to genome damage. They delay the cell cycle and activate repair mechanisms. The kinases at the heart of these pathways and the accessory proteins, which localize to DNA lesions and regulate kinase activation, are conserved from yeast to mammals. For Saccharomyces cerevisiae Rad9, a key adaptor protein in DNA damage checkpoint pathways, no clear human ortholog has yet been described in mammals. Rad9, however, shares localized homology with both human BRCA1 and 53BP1 since they all contain tandem C-terminal BRCT (BRCA1 C-terminal) motifs. 53BP1 is also a key mediator in DNA damage signaling required for cell cycle arrest, which has just been reported to possess a tandem Tudor repeat upstream of the BRCT motifs. Here we show that the major globular domain upstream of yeast Rad9 BRCT domains is structurally extremely similar to the Tudor domains recently resolved for 53BP1 and SMN. By expressing several fragments encompassing the Tudor-related motif and characterizing them using various physical methods, we isolated the independently folded unit for yeast Rad9. As in 53BP1, the domain corresponds to the SMN Tudor motif plus the contiguous HCA predicted structure region at the C terminus. These domains may help to further elucidate the structural and functional features of these two proteins and improve knowledge of the proteins involved in DNA damage.

The BRCT domain of mammalian Rev1 is involved in regulating DNA translesion synthesis

Rev1 is a deoxycytidyl transferase associated with DNA translesion synthesis (TLS). In addition to its catalytic domain, Rev1 possesses a so-called BRCA1 C-terminal (BRCT) domain. Here, we describe cells and mice containing a targeted deletion of this domain. Rev1B/B mice are healthy, fertile and display normal somatic hypermutation. Rev1B/B cells display an elevated spontaneous frequency of intragenic deletions at Hprt. In addition, these cells were sensitized to exogenous DNA damages. Ultraviolet-C (UV-C) light induced a delayed progression through late S and G2 phases of the cell cycle and many chromatid aberrations, specifically in a subset of mutant cells, but not enhanced sister chromatid exchanges (SCE). UV-C-induced mutagenesis was reduced and mutations at thymidine–thymidine dimers were absent in Rev1B/B cells, the opposite phenotype of UV-C-exposed cells from XP-V patients, lacking TLS polymerase η. This suggests that the enhanced UV-induced mutagenesis in XP-V patients may depend on error-prone Rev1-dependent TLS. Together, these data indicate a regulatory role of the Rev1 BRCT domain in TLS of a limited spectrum of endogenous and exogenous nucleotide damages during a defined phase of the cell cycle.

DNA damage-induced cell cycle checkpoint control requires CtIP, a phosphorylation-dependent binding partner of BRCA1 C-terminal domains.

The BRCA1 C-terminal (BRCT) domain has recently been implicated as a phospho-protein binding domain. We demonstrate here that a CTBP-interacting protein CtIP interacts with BRCA1 BRCT domains in a phosphorylation-dependent manner. The CtIP/BRCA1 complex only exists in G(2) phase and is required for DNA damage-induced Chk1 phosphorylation and the G(2)/M transition checkpoint. However, the CtIP/BRCA1 complex is not required for the damage-induced G(2) accumulation checkpoint, which is controlled by a separate BRCA1/BACH1 complex. Taken together, these data not only implicate CtIP as a critical player in cell cycle checkpoint control but also provide molecular mechanisms by which BRCA1 controls multiple cell cycle transitions after DNA damage.