BRCA2-HSF2BP oligomeric ring disassembly by BRME1 promotes homologous recombination.

In meiotic homologous recombination (HR), BRCA2 facilitates loading of the recombinases RAD51 and DMC1 at the sites of double-strand breaks (DSBs). The HSF2BP-BRME1 complex interacts with BRCA2. Its absence causes a severe reduction in recombinase loading at meiotic DSB. We previously showed that, in somatic cancer cells ectopically producing HSF2BP, DNA damage can trigger HSF2BP-dependent degradation of BRCA2, which prevents HR. Here, we report that, upon binding to BRCA2, HSF2BP forms octameric rings that are able to interlock into a large ring-shaped 24-mer. Addition of BRME1 leads to dissociation of both of these ring structures and cancels the disruptive effect of HSF2BP on cancer cell resistance to DNA damage. It also prevents BRCA2 degradation during interstrand DNA crosslink repair in Xenopus egg extracts. We propose that, during meiosis, the control of HSF2BPBRCA2 oligomerization by BRME1 ensures timely assembly of the ring complex that concentrates BRCA2 and controls its turnover, thus promoting HR.

G-quadruplex resolution: From molecular mechanisms to physiological relevance.

Guanine-rich DNA sequences can fold into stable four-stranded structures called G-quadruplexes or G4s. Research in the past decade demonstrated that G4 structures are widespread in the genome and prevalent in regulatory regions of actively transcribed genes. The formation of G4s has been tightly linked to important biological processes including regulation of gene expression and genome maintenance. However, they can also pose a serious threat to genome integrity especially by impeding DNA replication, and G4-associated somatic mutations have been found accumulated in the cancer genomes. Specialised DNA helicases and single stranded DNA binding proteins that can resolve G4 structures play a crucial role in preventing genome instability. The large variety of G4 unfolding proteins suggest the presence of multiple G4 resolution mechanisms in cells. Recently, there has been considerable progress in our detailed understanding of how G4s are resolved, especially during DNA replication. In this review, we first discuss the current knowledge of the genomic G4 landscapes and the impact of G4 structures on DNA replication and genome integrity. We then describe the recent progress on the mechanisms that resolve G4 structures and their physiological relevance. Finally, we discuss therapeutic opportunities to target G4 structures.

Efficient Synthesis of DNA Duplexes Containing Reduced Acetaldehyde Interstrand Cross-Links.

DNA interstrand cross-links (ICLs) prevent DNA replication and transcription and can lead to potentially lethal events, such as cancer or bone marrow failure. ICLs are typically repaired by proteins within the Fanconi Anemia (FA) pathway, although the details of the pathway are not fully established. Methods to generate DNA containing ICLs are key to furthering the understanding of DNA cross-link repair. A major route to ICL formation in vivo involves reaction of DNA with acetaldehyde, derived from ethanol metabolism. This reaction forms a three-carbon bridged ICL involving the amino groups of adjacent guanines in opposite strands of a duplex resulting in amino and imino functionalities. A stable reduced form of the ICL has applications in understanding the recognition and repair of these types of adducts. Previous routes to creating DNA duplexes containing these adducts have involved lengthy post-DNA synthesis chemistry followed by reduction of the imine. Here, an efficient and high-yielding approach to the reduced ICL using a novel N2-((R)-4-trifluoroacetamidobutan-2-yl)-2'-deoxyguanosine phosphoramidite is described. Following standard automated DNA synthesis and deprotection, the ICL is formed overnight in over 90% yield upon incubation at room temperature with a complementary oligodeoxyribonucleotide containing 2-fluoro-2'-deoxyinosine. The cross-linked duplex displayed a melting transition 25 °C higher than control sequences. Importantly, we show using the Xenopus egg extract system that an ICL synthesized by this method is repaired by the FA pathway. The simplicity and efficiency of this methodology for preparing reduced acetaldehyde ICLs will facilitate access to these DNA architectures for future studies on cross-link repair.

Sister chromatid exchanges induced by perturbed replication can form independently of BRCA1, BRCA2 and RAD51.

Sister chromatid exchanges (SCEs) are products of joint DNA molecule resolution, and are considered to form through homologous recombination (HR). Indeed, SCE induction upon irradiation requires the canonical HR factors BRCA1, BRCA2 and RAD51. In contrast, replication-blocking agents, including PARP inhibitors, induce SCEs independently of BRCA1, BRCA2 and RAD51. PARP inhibitor-induced SCEs are enriched at difficult-to-replicate genomic regions, including common fragile sites (CFSs). PARP inhibitor-induced replication lesions are transmitted into mitosis, suggesting that SCEs can originate from mitotic processing of under-replicated DNA. Proteomics analysis reveals mitotic recruitment of DNA polymerase theta (POLQ) to synthetic DNA ends. POLQ inactivation results in reduced SCE numbers and severe chromosome fragmentation upon PARP inhibition in HR-deficient cells. Accordingly, analysis of CFSs in cancer genomes reveals frequent allelic deletions, flanked by signatures of POLQ-mediated repair. Combined, we show PARP inhibition generates under-replicated DNA, which is processed into SCEs during mitosis, independently of canonical HR factors.

Alcohol-derived DNA crosslinks are repaired by two distinct mechanisms.

Acetaldehyde is a highly reactive, DNA-damaging metabolite that is produced upon alcohol consumption1. Impaired detoxification of acetaldehyde is common in the Asian population, and is associated with alcohol-related cancers1,2. Cells are protected against acetaldehyde-induced damage by DNA crosslink repair, which when impaired causes Fanconi anaemia (FA), a disease resulting in failure to produce blood cells and a predisposition to cancer3,4. The combined inactivation of acetaldehyde detoxification and the FA pathway induces mutation, accelerates malignancies and causes the rapid attrition of blood stem cells5-7. However, the nature of the DNA damage induced by acetaldehyde and how this is repaired remains a key question. Here we generate acetaldehyde-induced DNA interstrand crosslinks and determine their repair mechanism in Xenopus egg extracts. We find that two replication-coupled pathways repair these lesions. The first is the FA pathway, which operates using excision-analogous to the mechanism used to repair the interstrand crosslinks caused by the chemotherapeutic agent cisplatin. However, the repair of acetaldehyde-induced crosslinks results in increased mutation frequency and an altered mutational spectrum compared with the repair of cisplatin-induced crosslinks. The second repair mechanism requires replication fork convergence, but does not involve DNA incisions-instead the acetaldehyde crosslink itself is broken. The Y-family DNA polymerase REV1 completes repair of the crosslink, culminating in a distinct mutational spectrum. These results define the repair pathways of DNA interstrand crosslinks caused by an endogenous and alcohol-derived metabolite, and identify an excision-independent mechanism.

HSF2BP negatively regulates homologous recombination in DNA interstrand crosslink repair.

The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability and DNA interstrand crosslink (ICL) repair in vertebrates. We show that ectopic production of HSF2BP, a BRCA2-interacting protein required for meiotic HR during mouse spermatogenesis, in non-germline human cells acutely sensitize them to ICL-inducing agents (mitomycin C and cisplatin) and PARP inhibitors, resulting in a phenotype characteristic of cells from Fanconi anemia (FA) patients. We biochemically recapitulate the suppression of ICL repair and establish that excess HSF2BP compromises HR by triggering the removal of BRCA2 from the ICL site and thereby preventing the loading of RAD51. This establishes ectopic expression of a wild-type meiotic protein in the absence of any other protein-coding mutations as a new mechanism that can lead to an FA-like cellular phenotype. Naturally occurring elevated production of HSF2BP in tumors may be a source of cancer-promoting genomic instability and also a targetable vulnerability.

HSF2BP Interacts with a Conserved Domain of BRCA2 and Is Required for Mouse Spermatogenesis.

The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability, and DNA interstrand crosslink repair in vertebrates. We identify HSF2BP, a protein previously described as testis specific and not characterized functionally, as an interactor of BRCA2 in mouse embryonic stem cells, where the 2 proteins form a constitutive complex. HSF2BP is transcribed in all cultured human cancer cell lines tested and elevated in some tumor samples. Inactivation of the mouse Hsf2bp gene results in male infertility due to a severe HR defect during spermatogenesis. The BRCA2-HSF2BP interaction is highly evolutionarily conserved and maps to armadillo repeats in HSF2BP and a 68-amino acid region between the BRC repeats and the DNA binding domain of human BRCA2 (Gly2270-Thr2337) encoded by exons 12 and 13. This region of BRCA2 does not harbor known cancer-associated missense mutations and may be involved in the reproductive rather than the tumor-suppressing function of BRCA2.

Xenopus egg extract: A powerful tool to study genome maintenance mechanisms.

DNA repair pathways are crucial to maintain the integrity of our genome and prevent genetic diseases such as cancer. There are many different types of DNA damage and specific DNA repair mechanisms have evolved to deal with these lesions. In addition to these repair pathways there is an extensive signaling network that regulates processes important for repair, such as cell cycle control and transcription. Despite extensive research, DNA damage repair and signaling are not fully understood. In vitro systems such as the Xenopus egg extract system, have played, and still play, an important role in deciphering the molecular details of these processes. Xenopus laevis egg extracts contain all factors required to efficiently perform DNA repair outside a cell, using mechanisms conserved in humans. These extracts have been used to study several genome maintenance pathways, including mismatch repair, non-homologous end joining, ICL repair, DNA damage checkpoint activation, and replication fork stability. Here we describe how the Xenopus egg extract system, in combination with specifically designed DNA templates, contributed to our detailed understanding of these pathways.

The Fanconi anemia pathway promotes replication-dependent DNA interstrand cross-link repair.

Fanconi anemia is a human cancer predisposition syndrome caused by mutations in 13 Fanc genes. The disorder is characterized by genomic instability and cellular hypersensitivity to chemicals that generate DNA interstrand cross-links (ICLs). A central event in the activation of the Fanconi anemia pathway is the mono-ubiquitylation of the FANCI-FANCD2 complex, but how this complex confers ICL resistance remains enigmatic. Using a cell-free system, we showed that FANCI-FANCD2 is required for replication-coupled ICL repair in S phase. Removal of FANCD2 from extracts inhibits both nucleolytic incisions near the ICL and translesion DNA synthesis past the lesion. Reversal of these defects requires ubiquitylated FANCI-FANCD2. Our results show that multiple steps of the essential S-phase ICL repair mechanism fail when the Fanconi anemia pathway is compromised.

SUMO assay with peptide arrays on solid support: insights into SUMO target sites.

The modification of proteins by SUMO (small ubiquitin-like modifier) regulates various cellular processes. Sumoylation often occurs on a specific lysine residue within the consensus motif psiKxE/D. However, little is known about the specificity and selectivity of SUMO target sites. We describe here a SUMO assay with peptide array on solid support for the simultaneous characterization of hundreds of different SUMO target sites. This approach was used to characterize known SUMO substrates. The position of the motif within the peptide and the amino acids flanking the acceptor site affected the efficiency of SUMO modification. Interestingly, a sequence of only four amino acids, corresponding to the SUMO consensus motif without flanking amino acids, was a bona fide target site. Analysis of a peptide library for all variants of the psiKxE/D consensus motif revealed that the first and third positions in the tetrapeptide preferably contain aromatic amino acid residues. Furthermore, by adding the SUMO E3 ligase PIAS1 to the reaction mixture, we show specific enhancement of the modification of a PIAS1-dependent SUMO substrate in this system. Overall, our results demonstrate that the sumoylation assay with peptide array on solid support can be used for the high-throughput characterization of SUMO target sites, and provide new insights into the composition, selectivity and specificity of SUMO target sites.

Structure of the cyclic-AMP-responsive exchange factor Epac2 in its auto-inhibited state.

Epac proteins (exchange proteins directly activated by cAMP) are guanine-nucleotide-exchange factors (GEFs) for the small GTP-binding proteins Rap1 and Rap2 that are directly regulated by the second messenger cyclic AMP and function in the control of diverse cellular processes, including cell adhesion and insulin secretion. Here we report the three-dimensional structure of full-length Epac2, a 110-kDa protein that contains an amino-terminal regulatory region with two cyclic-nucleotide-binding domains and a carboxy-terminal catalytic region. The structure was solved in the absence of cAMP and shows the auto-inhibited state of Epac. The regulatory region is positioned with respect to the catalytic region by a rigid, tripartite beta-sheet-like structure we refer to as the 'switchboard' and an ionic interaction we call the 'ionic latch'. As a consequence of this arrangement, the access of Rap to the catalytic site is sterically blocked. Mutational analysis suggests a model for cAMP-induced Epac activation with rigid body movement of the regulatory region, the features of which are universally conserved in cAMP-regulated proteins.

Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2).

Multidrug resistance protein 2 (MRP2) belongs to the ATP binding cassette family of transporters. Its substrates include organic anions and anticancer drugs. We have used transport assays with vesicles derived from Sf9 insect cells overproducing MRP2 to study the interactions of drugs, organic anions, and bile acids with three MRP2 substrates: estradiol-17-beta-d-glucuronide (E217betaG), methotrexate, and glutathione-S-dinitrophenol. Complex inhibition and stimulation patterns were obtained, different from those observed with the related transporters MRP1 and MRP3. In contrast to a previous report, we found that the rate of E217betaG transport by MRP2 increases sigmoidally with substrate concentration indicative of homotropic cooperativity. Half-maximal transport was obtained at 120 microm E217betaG, in contrast to values < 20 microm for MRP1 and 3. MRP2 stimulators, such as indomethacin and sulfanitran, strongly increased the affinity of MRP2 for E217betaG (half-maximal transport rates at 65 and 16 microm E217betaG, respectively) and shifted the sigmoidal dependence of transport rate on substrate concentration to a more hyperbolic one, without substantially affecting the maximal transport rate. Sulfanitran also stimulated MRP2 activity in cells, i.e. the transport of saquinavir through monolayers of Madin-Darby canine kidney II cells. Some compounds that stimulate E217betaG transport, such as penicillin G or pantoprazole, are not detectably transported by MRP2, suggesting that they allosterically stimulate transport without being cotransported with E217betaG. We propose that MRP2 contains two similar but nonidentical ligand binding sites: one site from which substrate is transported and a second site that regulates the affinity of the transport site for the substrate.

High-throughput retroviral tagging to identify components of specific signaling pathways in cancer.

Genetic screens carried out in lower organisms such as yeast, Drosophila melanogaster and Caenorhabditis elegans have revealed many signaling pathways. For example, components of the RAS signaling cascade were identified using a mutant eye phenotype in D. melanogaster as a readout. Screening is usually based on enhancing or suppressing a phenotype by way of a known mutation in a particular signaling pathway. Such in vivo screens have been difficult to carry out in mammals, however, owing to their relatively long generation times and the limited number of animals that can be screened. Here we describe an in vivo mammalian genetic screen used to identify components of pathways contributing to oncogenic transformation. We applied retroviral insertional mutagenesis in Myc transgenic (E mu Myc) mice lacking expression of Pim1 and Pim2 to search for genes that can substitute for Pim1 and Pim2 in lymphomagenesis. We determined the chromosomal positions of 477 retroviral insertion sites (RISs) derived from 38 tumors from E mu Myc Pim1(-/-) Pim2(-/-) mice and 27 tumors from E mu Myc control mice using the Ensembl and Celera annotated mouse genome databases. There were 52 sites occupied by proviruses in more than one tumor. These common insertion sites (CISs) are likely to contain genes contributing to tumorigenesis. Comparison of the RISs in tumors of Pim-null mice with the RISs in tumors of E mu Myc control mice indicated that 10 of the 52 CISs belong to the Pim complementation group. In addition, we found that Pim3 is selectively activated in Pim-null tumor cells, which supports the validity of our approach.