Molecular architecture of the HerA–NurA DNA double-strand break resection complex

DNA double-strand breaks can be repaired by homologous recombination, during which the DNA ends are long-range resected by helicase–nuclease systems to generate 3′ single strand tails. In archaea, this requires the Mre11–Rad50 complex and the ATP-dependent helicase–nuclease complex HerA–NurA. We report the cryo-EM structure of Sulfolobus solfataricus HerA–NurA at 7.4 Å resolution and present the pseudo-atomic model of the complex. HerA forms an ASCE hexamer that tightly interacts with a NurA dimer, with each NurA protomer binding three adjacent HerA HAS domains. Entry to NurA’s nuclease active sites requires dsDNA to pass through a 23 Å wide channel in the HerA hexamer. The structure suggests that HerA is a dsDNA translocase that feeds DNA into the NurA nuclease sites.     

Function of the IsiA pigment–protein complex in vivo

Photosynthesis Research - The acclimation of cyanobacterial photosynthetic apparatus to iron deficiency is crucial for their performance under limiting conditions. In many cyanobacterial species,...     

Characterization of the peridinin–chlorophyll a-protein complex in the dinoflagellate Symbiodinium

The water-soluble peridinin–chlorophyll a-proteins (PCPs) are one of the major light harvesting complexes in photosynthetic dinoflagellates. PCP contains the carotenoid peridinin as its primary pigment. In this study, we identified and characterized the PCP protein and the PCP gene organization in Symbiodinium sp. CS-156. The protein molecular mass is 32.7 kDa, revealing that the PCP is of the monomeric form. The intronless PCP genes are organized in tandem arrays. The PCP gene cassette is composed of 1095-bp coding regions and spacers in between. Despite the heterogeneity of PCP gene tandem repeats, we identified a single form of PCP, the sequence of which exactly matches the deduced sequence of PCP gene clone 7 (JQ395030) by LC–MS/MS analysis of tryptic digested PCP, revealing the mature PCP apoprotein is 312 amino acids in length. Pigment analysis showed a peridinin-to-Chl a ratio of 4. The peridinin-to-Chl a Q_y energy transfer efficiency is 95% in this complex.     

Architecture of the Pol III–clamp–exonuclease complex reveals key roles of the exonuclease subunit in processive DNA synthesis and repair

DNA polymerase III (Pol III) is the catalytic α subunit of the bacterial DNA Polymerase III holoenzyme. To reach maximum activity, Pol III binds to the DNA sliding clamp β and the exonuclease ε that provide processivity and proofreading, respectively. Here, we characterize the architecture of the Pol III–clamp–exonuclease complex by chemical crosslinking combined with mass spectrometry and biochemical methods, providing the first structural view of the trimeric complex. Our analysis reveals that the exonuclease is sandwiched between the polymerase and clamp and enhances the binding between the two proteins by providing a second, indirect, interaction between the polymerase and clamp. In addition, we show that the exonuclease binds the clamp via the canonical binding pocket and thus prevents binding of the translesion DNA polymerase IV to the clamp, providing a novel insight into the mechanism by which the replication machinery can switch between replication, proofreading, and translesion synthesis.     

Electron microscopy of Xrcc4 and the DNA ligase IV–Xrcc4 DNA repair complex

The DNA ligase IV–Xrcc4 complex is responsible for the ligation of broken DNA ends in the non-homologous end-joining (NHEJ) pathway of DNA double strand break repair in mammals. Mutations in DNA ligase IV (Lig4) lead to immunodeficiency and radiosensitivity in humans. Only partial structural information for Lig4 and Xrcc4 is available, while the structure of the full-length proteins and their arrangement within the Lig4–Xrcc4 complex is unknown. The C-terminal domain of Xrcc4, whose structure has not been solved, contains phosphorylation sites for DNA-PKcs and is phylogenetically conserved, indicative of a regulatory role in NHEJ. Here, we have purified full length Xrcc4 and the Lig4–Xrcc4 complex, and analysed their structure by single-particle electron microscopy. The three-dimensional structure of Xrcc4 at a resolution of ～37 Å reveals that the C-terminus of Xrcc4 forms a dimeric globular domain connected to the N-terminus by a coiled-coil. The N- and C-terminal domains of Xrcc4 locate at opposite ends of an elongated molecule. The electron microscopy images of the Lig4–Xrcc4 complex were examined by two-dimensional image processing and a double-labelling strategy, identifying the site of the C-terminus of Xrcc4 and the catalytic core of Lig4 within the complex. The catalytic domains of Lig4 were found to be in the vicinity of the N-terminus of Xrcc4. We provide a first sight of the structural organization of the Lig4–Xrcc4 complex, which suggests that the BRCT domains could provide the link of the ligase to Xrcc4 while permitting some movements of the catalytic domains of Lig4. This arrangement may facilitate the ligation of diverse configurations of damaged DNA.     

The transformation of the cytoplasmic oestradiol–receptor complex into the nuclear complex in a uterine cell-free system

Experiments performed with a cell-free system in tris-EDTA buffer, pH 7.4, indicate that the high-speed supernatant fraction of the rat uterus contains all the factors necessary to transform the 8S cytoplasmic oestradiol-receptor complex to the nuclear complex. The transformation is temperature-dependent. This nuclear complex was extracted in the form of a 5S particle with 0.4m-KCl from sediments of either uterine or heart nuclei that had been incubated together with the cytoplasmic soluble fraction of the uterus at 2 degrees C for 30min. This complex can also be obtained similarly from the soluble fraction of the uterus, incubated in the absence of nuclei. Previous warming of the soluble fraction to 37 degrees C for 7min was necessary for the successful extraction of the nuclear particle under these conditions of incubation. After an incubation of the transformed complex with the nuclear sediment at 37 degrees C for 7min, the 5S complex was extractable from the uterine nuclear sediment but not from the heart nuclear sediment, which may indicate the tissue specificity of the nuclear acceptor sites for the transformed complex. The extracted uterine nuclear complex sediments in the 5S region, but whether it is the native complex or a subunit or other part of the native complex resulting from the extraction with salt is unknown.     

DNA–DNA cross-linking mediated by bifunctional [SalenAl] complex

The aluminum (III) complex [SalenAl^III]Cl (1), (Salen = (R,R)-N,N′-bis[5-methyl-3-(4-methylpiperazinyl)-salicylidene]-1,2-diphenylethanediamine) has been synthesized and characterized by elemental analysis, FT-IR, ¹H and ¹³C NMR measurements. The interaction of complex (1) with calf thymus (CT) DNA has been studied extensively by experimental techniques. Thermal denaturation study of DNA with (1) revealed the ΔT_m of 5 ± 0.2 °C. Viscosity and steady-state fluorescence measurements showed that the complex cross-links DNA and the metal center is interacting with DNA during the cross-linking. Also, the phenyl ring in the complex may intercalate between the base pairs of the DNA during the cross-linking. Competitive binding study shows that the enhanced emission intensity of ethidium bromide (EB) in the presence of DNA was quenched by the addition of the metal complex indicating that it displaces EB from its binding site in DNA and the apparent binding constant has been estimated to be (2.8 ± 0.2) × 10⁵ M^− 1. Further, time-resolved fluorescence experiments confirm the binding of (1) with DNA and its cross-linking nature. Aluminum ions shown to precipitate DNA completely above the pH 6.0, but no such precipitation was observed with complex (1). The DNA–DNA cross-linking mediated by (1) is further confirmed by gel electrophoresis.     

Advances in the study of protein–DNA interaction

Protein-DNA interaction plays an important role in many biological processes. The classical methods and the novel technologies advanced have been developed for the interaction of protein-DNA. Recent developments of these methods and research achievements have been reviewed in this paper.     

Mitochondrial dysfunction in cardiac ischemia–reperfusion injury: ROS from complex I,without inhibition

A key pathologic event in cardiac ischemia reperfusion (I–R) injury is mitochondrial energetic dysfunction, and several studies have attributed this to complex I (CxI) inhibition. In isolated perfused rat hearts, following I–R, we found that CxI-linked respiration was inhibited, but isolated CxI enzymatic activity was not. Using the mitochondrial thiol probe iodobutyl-triphenylphosphonium in conjunction with proteomic tools, thiol modifications were identified in several subunits of the matrix-facing 1α sub-complex of CxI. These thiol modifications were accompanied by enhanced ROS generation from CxI, but not complex III. Implications for the pathology of cardiac I–R injury are discussed.     

Mitochondrial DNA divergence in yoshinobori gobies (Rhinogobius species complex) between the Bonin Islands and the Japan–Ryukyu Archipelago

To investigate the genetic differentiation between the Bonin (Ogasawara) Islands' freshwater goby Rhinogobius sp. Bonin Island (BI) form (Ogasawara-yoshinobori) and the Japan–Ryukyu Archipelago relatives, the mitochondrial DNA (mtDNA) phylogeny of Japanese Rhinogobius species was inferred from partial nucleotide sequences of the mitochondrial NADH dehydrogenase 5 subunit (ND5) gene (945 bp). The resultant tree showed that the Bonin Islands group separated first from the other Japanese lineage, and a test calculation indicated the divergence date to be approximately 3 million years BP. Although it is necessary to use a more reliable estimate to confirm the divergence date, Rhinogobius sp. BI has retained its mtDNA lineage in the islands for millions of years.     

The DNA–DNA spacing in gemini surfactants–DOPE–DNA complexes

Gemini surfactants from the homologous series of alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide) (CnCS12, number of spacer carbons n = 2 – 12) and dioleoylphosphatidylethanolamine (DOPE) were used for cationic liposome (CL) preparation. CLs condense highly polymerized DNA creating complexes. Small-angle X-ray diffraction identified them as condensed lamellar phase L_α^C in the studied range of molar ratios CnGS12/DOPE in the temperature range 20 – 60 °C. The DNA–DNA distance (d_DNA) is studied in dependence to CnGS12 spacer length and membrane surface charge density. The high membrane surface charge densities (CnGS12/DOPE = 0.35 and 0.4 mol/mol) lead to the linear dependence of d_DNA vs. n correlating with the interfacial area of the CnGS12 molecule.     

Compensatory mutations in the L30e kink-turn RNA–protein complex

The S. cerevisiae ribosomal protein L30e is an autoregulatory protein that binds to its own pre-mRNA and mature mRNA to inhibit splicing and translation, respectively. The L30e RNA-binding element is a stem-asymmetric loop–stem that forms a kink-turn. A bacterial genetic system was designed to test the ability of protein variants to repress the expression of reporter mRNAs containing the L30e RNA-binding element. Initial screens revealed that changes in several RNA nucleotides had a measurable effect on repression of the reporter by the wild type protein. RNA mutants that reduce repression were screened against libraries of randomly mutagenized L30e proteins. These screens identified a glycine to serine mutation of L30e, which specifically restores activity to an RNA variant containing a U that replaces a helix-capping G. Similarly, an asparagine to alanine mutation was found to suppress a substitution at a position where the L30e RNA nucleotide extends out into the protein pocket. In addition, a compensatory RNA mutation within a defective RNA variant was found. The identification of these suppressors provides new insights into the architecture of a functional binding element and its recognition by an important RNA-binding protein.     

Phylogenetic structure of the Thomomys bottae–umbrinus complex in North America

The phylogeography of the Thomomys bottae–umbrinus complex in the United States and Mexico was assessed with sequences of the mitochondrial cytochrome b gene. These sequences were obtained from 225 individuals representing 108 locations over the range, including 56 sequences from GenBank. 110 (500 bp) sequences were used for Bayesian inference and neighbor-joining analyses, and 34 (1140 bp) specimens from the main clades obtained from the Bayesian inference were used in maximum-parsimony and maximum-likelihood analyses. The different analyses indicate significant variation within the species complex that averages 13% among major groups of genetic differences among Thomomys bottae–umbrinus. The overall pattern of geographic variation is not concordant with the current taxonomy. To the contrary, eight monophyletic groups are supported by all analyses and can be considered phylogenetic species. Overall divergence among these groups appears influenced by historical biogeographic events active during the Pliocene and Pleistocene.     

Structure of the UreD–UreF–UreG–UreE complex in Helicobacter pylori: a model study

The molecular details of the protein complex formed by UreD, UreF, UreG, and UreE, accessory proteins for urease activation in the carcinogenic bacterium Helicobacter pylori, have been elucidated using computational modeling. The calculated structure of the complex supports the hypothesis of UreF acting as a GTPase activation protein that facilitates GTP hydrolysis by UreG during urease maturation, and provides a rationale for the design of new drugs against infections by ureolytic bacterial pathogens.