Cryo-EM structure of the Mre11-Rad50-Nbs1 complex reveals the molecular mechanism of scaffolding functions.

The DNA double-strand break repair complex Mre11-Rad50-Nbs1 (MRN) detects and nucleolytically processes DNA ends, activates the ATM kinase, and tethers DNA at break sites. How MRN can act both as nuclease and scaffold protein is not well understood. The cryo-EM structure of MRN from Chaetomium thermophilum reveals a 2:2:1 complex with a single Nbs1 wrapping around the autoinhibited Mre11 nuclease dimer. MRN has two DNA-binding modes, one ATP-dependent mode for loading onto DNA ends and one ATP-independent mode through Mre11's C terminus, suggesting how it may interact with DSBs and intact DNA. MRNs two 60-nm-long coiled-coil domains form a linear rod structure, the apex of which is assembled by the two joined zinc-hook motifs. Apices from two MRN complexes can further dimerize, forming 120-nm spanning MRN-MRN structures. Our results illustrate the architecture of MRN and suggest how it mechanistically integrates catalytic and tethering functions.

Mechanism of DNA End Sensing and Processing by the Mre11-Rad50 Complex.

DNA double-strand breaks (DSBs) threaten genome stability throughout life and are linked to tumorigenesis in humans. To initiate DSB repair by end joining or homologous recombination, the Mre11-nuclease Rad50-ATPase complex detects and processes diverse and obstructed DNA ends, but a structural mechanism is still lacking. Here we report cryo-EM structures of the E. coli Mre11-Rad50 homolog SbcCD in resting and DNA-bound cutting states. In the resting state, Mre11's nuclease is blocked by ATP-Rad50, and the Rad50 coiled coils appear flexible. Upon DNA binding, the two coiled coils zip up into a rod and, together with the Rad50 nucleotide-binding domains, form a clamp around dsDNA. Mre11 moves to the side of Rad50, binds the DNA end, and assembles a DNA cutting channel for the nuclease reactions. The structures reveal how Mre11-Rad50 can detect and process diverse DNA ends and uncover a clamping and gating function for the coiled coils.

Phage dUTPases control transfer of virulence genes by a proto-oncogenic G protein-like mechanism.

dUTPases (Duts) have emerged as promising regulatory molecules controlling relevant cellular processes. However, the mechanism underlying this regulatory function remains enigmatic. Using staphylococcal pathogenicity island (SaPI) repression as a model, we report here that phage Duts induce the transfer of SaPI-encoded virulence factors by switching between active (dUTP-bound) and inactive (apo state) conformations, a conversion catalyzed by their intrinsic dUTPase activity. Crystallographic and mutagenic analyses demonstrate that binding to dUTP reorders the C-terminal motif V of the phage-encoded Duts, rendering these proteins into the active conformation required for SaPI derepression. By contrast, the conversion to the apo state conformation by hydrolysis of the bound dUTP generates a protein that is unable to induce the SaPI cycle. Because none of the requirements involving Duts in SaPI transfer are exclusive to the phage-encoded proteins, we propose that Duts are widespread cellular regulators acting in a manner analogous to the eukaryotic G proteins.

Chromatin association of the SMC5/6 complex is dependent on binding of its NSE3 subunit to DNA.

SMC5/6 is a highly conserved protein complex related to cohesin and condensin, which are the key components of higher-order chromatin structures. The SMC5/6 complex is essential for proliferation in yeast and is involved in replication fork stability and processing. However, the precise mechanism of action of SMC5/6 is not known. Here we present evidence that the NSE1/NSE3/NSE4 sub-complex of SMC5/6 binds to double-stranded DNA without any preference for DNA-replication/recombination intermediates. Mutations of key basic residues within the NSE1/NSE3/NSE4 DNA-binding surface reduce binding to DNA in vitro. Their introduction into the Schizosaccharomyces pombe genome results in cell death or hypersensitivity to DNA damaging agents. Chromatin immunoprecipitation analysis of the hypomorphic nse3 DNA-binding mutant shows a reduced association of fission yeast SMC5/6 with chromatin. Based on our results, we propose a model for loading of the SMC5/6 complex onto the chromatin.

Structural analysis of the PATZ1 BTB domain homodimer.

PATZ1 is a ubiquitously expressed transcriptional repressor belonging to the ZBTB family that is functionally expressed in T lymphocytes. PATZ1 targets the CD8 gene in lymphocyte development and interacts with the p53 protein to control genes that are important in proliferation and in the DNA-damage response. PATZ1 exerts its activity through an N-terminal BTB domain that mediates dimerization and co-repressor interactions and a C-terminal zinc-finger motif-containing domain that mediates DNA binding. Here, the crystal structures of the murine and zebrafish PATZ1 BTB domains are reported at 2.3 and 1.8 Šresolution, respectively. The structures revealed that the PATZ1 BTB domain forms a stable homodimer with a lateral surface groove, as in other ZBTB structures. Analysis of the lateral groove revealed a large acidic patch in this region, which contrasts with the previously resolved basic co-repressor binding interface of BCL6. A large 30-amino-acid glycine- and alanine-rich central loop, which is unique to mammalian PATZ1 amongst all ZBTB proteins, could not be resolved, probably owing to its flexibility. Molecular-dynamics simulations suggest a contribution of this loop to modulation of the mammalian BTB dimerization interface.

Specialized interfaces of Smc5/6 control hinge stability and DNA association.

The Structural Maintenance of Chromosomes (SMC) complexes: cohesin, condensin and Smc5/6 are involved in the organization of higher-order chromosome structure-which is essential for accurate chromosome duplication and segregation. Each complex is scaffolded by a specific SMC protein dimer (heterodimer in eukaryotes) held together via their hinge domains. Here we show that the Smc5/6-hinge, like those of cohesin and condensin, also forms a toroidal structure but with distinctive subunit interfaces absent from the other SMC complexes; an unusual 'molecular latch' and a functional 'hub'. Defined mutations in these interfaces cause severe phenotypic effects with sensitivity to DNA-damaging agents in fission yeast and reduced viability in human cells. We show that the Smc5/6-hinge complex binds preferentially to ssDNA and that this interaction is affected by both 'latch' and 'hub' mutations, suggesting a key role for these unique features in controlling DNA association by the Smc5/6 complex.

Destabilized SMC5/6 complex leads to chromosome breakage syndrome with severe lung disease.

The structural maintenance of chromosomes (SMC) family of proteins supports mitotic proliferation, meiosis, and DNA repair to control genomic stability. Impairments in chromosome maintenance are linked to rare chromosome breakage disorders. Here, we have identified a chromosome breakage syndrome associated with severe lung disease in early childhood. Four children from two unrelated kindreds died of severe pulmonary disease during infancy following viral pneumonia with evidence of combined T and B cell immunodeficiency. Whole exome sequencing revealed biallelic missense mutations in the NSMCE3 (also known as NDNL2) gene, which encodes a subunit of the SMC5/6 complex that is essential for DNA damage response and chromosome segregation. The NSMCE3 mutations disrupted interactions within the SMC5/6 complex, leading to destabilization of the complex. Patient cells showed chromosome rearrangements, micronuclei, sensitivity to replication stress and DNA damage, and defective homologous recombination. This work associates missense mutations in NSMCE3 with an autosomal recessive chromosome breakage syndrome that leads to defective T and B cell function and acute respiratory distress syndrome in early childhood.

Structural and functional insights into endoglin ligand recognition and binding.

Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascular endothelial cells, is a component of the transforming growth factor (TGF)-ß receptor complex and is implicated in a dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia as well as in preeclampsia. It interacts with the type I TGF-ß signaling receptor activin receptor-like kinase (ALK)1 and modulates cellular responses to Bone Morphogenetic Protein (BMP)-9 and BMP-10. Structurally, besides carrying a zona pellucida (ZP) domain, endoglin contains at its N-terminal extracellular region a domain of unknown function and without homology to any other known protein, therefore called the orphan domain (OD). In this study, we have determined the recognition and binding ability of full length ALK1, endoglin and constructs encompassing the OD to BMP-9 using combined methods, consisting of surface plasmon resonance and cellular assays. ALK1 and endoglin ectodomains bind, independently of their glycosylation state and without cooperativity, to different sites of BMP-9. The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition. These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin. In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted.

Mechanism of transcriptional stalling at cisplatin-damaged DNA.

The anticancer drug cisplatin forms 1,2-d(GpG) DNA intrastrand cross-links (cisplatin lesions) that stall RNA polymerase II (Pol II) and trigger transcription-coupled DNA repair. Here we present a structure-function analysis of Pol II stalling at a cisplatin lesion in the DNA template. Pol II stalling results from a translocation barrier that prevents delivery of the lesion to the active site. AMP misincorporation occurs at the barrier and also at an abasic site, suggesting that it arises from nontemplated synthesis according to an 'A-rule' known for DNA polymerases. Pol II can bypass a cisplatin lesion that is artificially placed beyond the translocation barrier, even in the presence of a G.A mismatch. Thus, the barrier prevents transcriptional mutagenesis. The stalling mechanism differs from that of Pol II stalling at a photolesion, which involves delivery of the lesion to the active site and lesion-templated misincorporation that blocks transcription.

Bypass of DNA lesions generated during anticancer treatment with cisplatin by DNA polymerase eta.

DNA polymerase eta (Pol eta) is a eukaryotic lesion bypass polymerase that helps organisms to survive exposure to ultraviolet (UV) radiation, and tumor cells to gain resistance against cisplatin-based chemotherapy. It allows cells to replicate across cross-link lesions such as 1,2-d(GpG) cisplatin adducts (Pt-GG) and UV-induced cis-syn thymine dimers. We present structural and biochemical analysis of how Pol eta copies Pt-GG-containing DNA. The damaged DNA is bound in an open DNA binding rim. Nucleotidyl transfer requires the DNA to rotate into an active conformation, driven by hydrogen bonding of the templating base to the dNTP. For the 3'dG of the Pt-GG, this step is accomplished by a Watson-Crick base pair to dCTP and is biochemically efficient and accurate. In contrast, bypass of the 5'dG of the Pt-GG is less efficient and promiscuous for dCTP and dATP as a result of the presence of the rigid Pt cross-link. Our analysis reveals the set of structural features that enable Pol eta to replicate across strongly distorting DNA lesions.

Natural ozone scavenger prevents asthma in sensitized rats.

The assumption that ozone is not only a strong oxidant, but also an important inflammatory mediator, is heavily supported by the ample literature on the pulmonary toxicity and biological effects of environmental ozone and by the recent discovery that antibodies, human neutrophils, and inflammatory lesions catalyze the formation of ozone in vivo. We hypothesized that the pulmonary inflammation in asthma involves a vicious circle of ozone production and recruitment of white blood cells, which produce more ozone. Accordingly, we predicted that electron-rich olefins, which are known ozone scavengers, could be used for prophylactic treatment of asthma. In particular, volatile, unsaturated monoterpenes, could saturate the pulmonary membranes and thereby equip the airways with local chemical protection against either exogenous or endogenous ozone. Here we present experimental evidence using a sensitized rat model to support this hypothesis. Examination of the pulmonary function of sensitized rats that inhaled either limonene (unsaturated, ozone scavenger) or eucalyptol (saturated, inert to ozone) showed that limonene inhalation significantly prevents bronchial obstruction while eucalyptol inhalation does not cause any effect. The anti-inflammatory effect of limonene was also evident from pathological parameters, such as diminished peribronchiolar and perivascular inflammatory infiltrates.

Decomposition of triacetone triperoxide is an entropic explosion.

Both X-ray crystallography and electronic structure calculations using the cc-pVDZ basis set at the DFT B3LYP level were employed to study the explosive properties of triacetone triperoxide (TATP) and diacetone diperoxide (DADP). The thermal decomposition pathway of TATP was investigated by a series of calculations that identified transition states, intermediates, and the final products. Counterintuitively, these calculations predict that the explosion of TATP is not a thermochemically highly favored event. It rather involves entropy burst, which is the result of formation of one ozone and three acetone molecules from every molecule of TATP in the solid state.