Brca1 C-terminal (BRCT) domains are a common protein-protein interaction motif in proteins involved in the DNA damage response and DNA repair. The DNA-damage response protein 53BP1 has two BRCT domains that bind to the DNA-binding domain of p53. The 53BP1 tandem-BRCT region is homologous to the tandem-BRCT region of Brca1, which is involved in double-strand break repair and homologous recombination and which binds BACH1, a member of the DEAH helicase family. Here we report the structures of a human 53BP1-p53 complex and of the rat Brca1 BRCT repeats. The 53BP1-p53 structure shows that the two BRCT repeats are arranged tandemly and pack extensively through an interface that also involves the inter-repeat linker. The first BRCT repeat and the linker together bind p53 on a region that overlaps with the DNA-binding surface of p53 and involves p53 residues that are mutated in cancer and are important for DNA binding. Comparison with the structure of the tandem-BRCT region of Brca1 shows a remarkable conservation of the repeat arrangement and of the inter-BRCT repeat interface. Analysis of human BRCA1 tumor-derived mutations and conservation identifies a potential protein-binding site that we show through mutagenesis is involved in BACH1 binding. The BACH1-binding region of Brca1 consists of a unique insertion in the first BRCT repeat and the inter-repeat linker and is analogous to the region of 53BP1 that binds p53.

Fanconi Anemia is a cancer predisposition syndrome caused by defects in the repair of DNA interstrand crosslinks (ICL). Central to this pathway is the FANCI-FANCD2 (ID) complex, which is activated by DNA damage-induced phosphorylation and monoubiquitination. The 3.4 Å crystal structure of the ~300 kDa ID complex reveals that monoubiquitination and regulatory phosphorylation sites map to the I-D interface, suggesting that they occur on monomeric proteins or an opened-up complex, and that they may serve to stabilize I-D hetero-dimerization. The 7.8 Å electron density map of FANCI-DNA crystals and in vitro data show that each protein has binding sites for both single- and double-stranded DNA, suggesting that the ID complex recognizes DNA structures that result from the encounter of replication forks with an ICL.

The tumor suppressor gene p53 in mammalian cells plays a critical role in safeguarding the integrity of genome. It functions as a sequence-specific transcription factor. Upon activation by a variety of cellular stresses, p53 transactivates downstream target genes, through which it regulates cell cycle and apoptosis. However, little is known about p53 in invertebrates. Here we report the identification and characterization of a Drosophila p53 homologue gene, dp53. dp53 encodes a 385-amino acid protein with significant homology to human p53 (hp53) in the region of the DNA-binding domain, and to a lesser extent the tetramerization domain. Purified dp53 DNA-binding domain protein was shown to bind to the consensus hp53-binding site by gel mobility analysis. In transient transfection assays, expression of dp53 in Schneider cells transcriptionally activated promoters that contained consensus hp53-responsive elements. Moreover, a mutant dp53 (Arg-155 to His-155), like its hp53 counterpart mutant, exerted a dominant-negative effect on transactivation. Ectopic expression of dp53 in Drosophila eye disk caused cell death and led to a rough eye phenotype. dp53 is expressed throughout the development of Drosophila with highest expression levels in early embryogenesis, which has a maternal component. Consistent with this, dp53 RNA levels were high in the nurse cells of the ovary. It appears that p53 is structurally and functionally conserved from flies to mammals. Drosophila will provide a useful genetic system to the further study of the p53 network.

The BRCT (BRCA1 C-terminus) is an evolutionary conserved protein±protein interacting module found as single, tandem or multiple repeats in a diverse range of proteins known to play roles in the DNAdamage response. The BRCT domains of 53BP1 bind to the tumour suppressor p53. To investigate the nature of this interaction, we have determined the crystal structure of the 53BP1 BRCT tandem repeat in complex with the DNA-binding domain of p53. The structure of the 53BP1±p53 complex shows that the BRCT tandem repeats pack together through a conserved interface that also involves the inter-domain linker. A comparison of the structure of the BRCT region of 53BP1 with the BRCA1 BRCT tandem repeat reveals that the interdomain interface and linker regions are remarkably well conserved. 53BP1 binds to p53 through contacts with the N-terminal BRCT repeat and the inter-BRCT linker. The p53 residues involved in this binding are mutated in cancer and are also important for DNA binding. We propose that BRCT domains bind to cellular target proteins through a conserved structural element termed the`BRCT recognition motif'.

Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag)hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on "active pairs" of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the fourpentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.The protein-DNA interactions that take place at eukaryotic origins of DNA replication are poorly characterized. This situation reflects, in part, the failure to identify DNA sequences that constitute higher eukaryotic origins of replication (13). Moreover, there is limited structural information about the initiator proteins that recognize origins of replication (35, 67). Indeed, structural information is currently limited to the origin binding domains of initiators encoded by simian virus 40 (SV40) (45), bovine papillomavirus (33), and Epstein-Barr virus (1, 2).In view of these limitations, a useful model for studies of the protein-DNA interactions that take place at eukaryotic origins is the binding of the virus-encoded T-antigen (T-ag) to the SV40 origin of replication. The well-characterized SV40 core origin is 64 bp long and consists of three separate domains (20,21). The central region, termed site II (or pentanucleotide palindrome), contains four GAGGC pentanucleotides that serve as binding sites for T-ag (24, 69, 70). Site II is flanked by a 17-bp adenine-thymine (AT)-rich domain and the early palindrome (EP) (reviewed in references 4 and 29).T-ag, a 708-amino-acid phosphoprotein, has been extensively studied (reviewed in references 4, 9, and 29). The structure of the T-ag domain that is necessary and sufficient for binding to the SV40 origin, T-ag-obd , was solved by use of nuclear magnetic resonance techniques (45). When the structure was viewed in terms of previous mutagenesis studies of T-ag (65, 76), considerable insight into the mechanism of binding of T-ag to individual pentanucleotides was obtained. For example, it is now apparent that site-specific binding is mediated by a pair of loops (45), a common motif in protein-DNA interactions (9, 41). Additional insights into T-ag binding to individual pentanucleotides, ...

To better define protein-DNA interactions at a eukaryotic origin, the domain of simian virus 40 (SV40) large T antigen that specifically interacts with the SV40 origin has been purified and its binding to DNA has been characterized. Evidence is presented that the affinity of the purified T antigen DNA-binding domain for the SV40 origin is comparable to that of the full-length T antigen. Furthermore, stable binding of the T antigen DNA-binding domain to the SV40 origin requires pairs of pentanucleotide recognition sites separated by approximately one turn of a DNA double helix and positioned in a head-to-head orientation. Although two pairs of pentanucleotides are present in the SV40 origin, footprinting and band shift experiments indicate that binding is limited to dimer formation on a single pair of pentanucleotides. Finally, it is demonstrated that the T antigen DNA-binding domain interacts poorly with single-stranded DNA.

Replication initiation events are suppressed over the SV40 core origin in vitro; they are also greatly reduced over sequences flanking the origin which contain binding sites for several transcription factors. To address the biochemical basis for the gap in initiation events over the flanking sequences, initial synthesis events have been characterized on templates lacking these sequences. Herein, it is demonstrated that previously functional initiation sites are nearly inactive when moved to positions that are proximal to the core origin. Thus, the gap in initiation events depends, in part, on the proximity of the initiation sites to the SV40 core origin. Additional experiments demonstrate that removal of the flanking sequences had little or no effect on DNA unwinding or on the efficiency of initiation of DNA synthesis in vitro. These results indicate that, under our in vitro conditions, initiation of SV40 DNA synthesis is not enhanced by binding of transcription factors to the flanking sequences.

The regions of the simian virus 40 (SV40) core origin that are required for stable assembly of virally encoded T antigen (T-ag) and the T-ag origin binding domain (T-ag-obd131–260) have been determined. Binding of the purified T-ag-obd131–260 is mediated by interactions with the central region of the core origin, site II. In contrast, T-ag binding and hexamer assembly requires a larger region of the core origin that includes both site II and an additional fragment of DNA that may be positioned on either side of site II. These studies indicate that in the context of T-ag, the origin binding domain can engage the pentanucleotides in site II only if a second region of T-ag interacts with one of the flanking sequences. The requirements for T-ag double-hexamer assembly are complex; the nucleotide cofactor present in the reaction modulates the sequence requirements for oligomerization. Nevertheless, these experiments provide additional evidence that only a subset of the SV40 core origin is required for assembly of T-ag double hexamers.