The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair

The Mre11 complex (Mre11 Rad50 Nbs1) is central to chromosomal maintenance and functions in homologous recombination, telomere maintenance and sister chromatid association. These functions all imply that the linked binding of two DNA substrates occurs, although the molecular basis for this process remains unknown. Here we present a 2.2 A crystal structure of the Rad50 coiled-coil region that reveals an unexpected dimer interface at the apex of the coiled coils in which pairs of conserved Cys-X-X-Cys motifs form interlocking hooks that bind one Zn(2+) ion. Biochemical, X-ray and electron microscopy data indicate that these hooks can join oppositely protruding Rad50 coiled-coil domains to form a flexible bridge of up to 1,200 A. This suggests a function for the long insertion in the Rad50 ABC-ATPase domain. The Rad50 hook is functional, because mutations in this motif confer radiation sensitivity in yeast and disrupt binding at the distant Mre11 nuclease interface. These data support an architectural role for the Rad50 coiled coils in forming metal-mediated bridging complexes between two DNA-binding heads. The resulting assemblies have appropriate lengths and conformational properties to link sister chromatids in homologous recombination and DNA ends in non-homologous end-joining.     

Structural insight into filament formation by mammalian septins

Septins are GTP-binding proteins that assemble into homo- and hetero-oligomers and filaments. Although they have key roles in various cellular processes, little is known concerning the structure of septin subunits or the organization and polarity of septin complexes. Here we present the structures of the human SEPT2 G domain and the heterotrimeric human SEPT2-SEPT6-SEPT7 complex. The structures reveal a universal bipolar polymer building block, composed of an extended G domain, which forms oligomers and filaments by conserved interactions between adjacent nucleotide-binding sites and/or the amino- and carboxy-terminal extensions. Unexpectedly, X-ray crystallography and electron microscopy showed that the predicted coiled coils are not involved in or required for complex and/or filament formation. The asymmetrical heterotrimers associate head-to-head to form a hexameric unit that is nonpolarized along the filament axis but is rotationally asymmetrical. The architecture of septin filaments differs fundamentally from that of other cytoskeletal structures.     

CCR5膜外二硫键Cys20-Cys269对其构象完整性以及功能行使起关键作用

人类化学趋化因子受体CCR5是HIV-1病毒进入人体细胞的主要辅助受体。实验表明,CCR5分子的N末端区域对化学趋化因子结合以及HIV病毒入侵起关键作用。用同源模建的方法构建CCR5结构模型,并对该模型的胞外结构域进行了1ns高温分子动力学和200ps常温分子动力学模拟。结果表明,CCR5的胞外结构域整体上表现为一个包装紧密的球形构象,二硫键Cys20-Cys269对这一构象的形成以及N末端区域的空间取向起关键作用,同时,二硫键的存在使N末端1-19区域定位在胞外结构域的顶部,并增加N末端构象柔性,使其有机会伸展到胞外空间去。根据这一结果和现有的实验证据,提出了HIV-CCR5两步结合机制。     

TRAPPI tethers COPII vesicles by binding the coat subunit Sec23

The budding of endoplasmic reticulum (ER)-derived vesicles is dependent on the COPII coat complex. Coat assembly is initiated when Sar1-GTP recruits the cargo adaptor complex, Sec23/Sec24, by binding to its GTPase-activating protein (GAP) Sec23 (ref. 2). This leads to the capture of transmembrane cargo by Sec24 (refs 3, 4) before the coat is polymerized by the Sec13/Sec31 complex. The initial interaction of a vesicle with its target membrane is mediated by tethers. We report here that in yeast and mammalian cells the tethering complex TRAPPI (ref. 7) binds to the coat subunit Sec23. This event requires the Bet3 subunit. In vitro studies demonstrate that the interaction between Sec23 and Bet3 targets TRAPPI to COPII vesicles to mediate vesicle tethering. We propose that the binding of TRAPPI to Sec23 marks a coated vesicle for fusion with another COPII vesicle or the Golgi apparatus. An implication of these findings is that the intracellular destination of a transport vesicle may be determined in part by its coat and its associated cargo.     

Reciprocal regulation of CD4/CD8 expression by SWI/SNF-like BAF complexes

Thymic development produces two sub-lineages of T cells expressing either CD4 or CD8 co-receptors that assist antibody production and mediate cell killing, respectively. The mechanisms for mutually exclusive co-receptor expression remain poorly defined. We find that mutations in the high mobility group (HMG) domain of BAF57--a DNA-binding subunit of the mammalian SWI/SNF-like chromatin-remodelling BAF complexes--or in the BAF complex ATPase subunit Brg, impair both CD4 silencing and CD8 activation. Brg is haploinsufficient for CD8 activation, but not for CD4 silencing, whereas BAF57 mutations preferentially impair CD4 silencing, pointing to target- and subunit-specific mechanisms of chromatin remodelling. BAF complexes directly bind the CD4 silencer, but the BAF57 HMG domain is dispensable for tethering BAF complexes to the CD4 silencer or other chromatin loci in vivo, or for remodelling reconstituted templates in vitro, suggesting that chromatin remodelling in vivo requires HMG-dependent DNA bending. These results indicate that BAF complexes contribute to lineage bifurcation by reciprocally regulating lineage-specific genes, reminiscent of the role of the yeast SWI/SNF complex in mediating mating-type switching.     

Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus

The critical viral components for packaging DNA, recognizing and binding to host cells, and injecting the condensed DNA into the host are organized at a single vertex of many icosahedral viruses. These component structures do not share icosahedral symmetry and cannot be resolved using a conventional icosahedral averaging method. Here we report the structure of the entire infectious Salmonella bacteriophage epsilon15 (ref. 1) determined from single-particle cryo-electron microscopy, without icosahedral averaging. This structure displays not only the icosahedral shell of 60 hexamers and 11 pentamers, but also the non-icosahedral components at one pentameric vertex. The densities at this vertex can be identified as the 12-subunit portal complex sandwiched between an internal cylindrical core and an external tail hub connecting to six projecting trimeric tailspikes. The viral genome is packed as coaxial coils in at least three outer layers with approximately 90 terminal nucleotides extending through the protein core and the portal complex and poised for injection. The shell protein from icosahedral reconstruction at higher resolution exhibits a similar fold to that of other double-stranded DNA viruses including herpesvirus, suggesting a common ancestor among these diverse viruses. The image reconstruction approach should be applicable to studying other biological nanomachines with components of mixed symmetries.     

Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism

DNA methylation of CpG dinucleotides is an important epigenetic modification of mammalian genomes and is essential for the regulation of chromatin structure, of gene expression and of genome stability. Differences in DNA methylation patterns underlie a wide range of biological processes, such as genomic imprinting, inactivation of the X chromosome, embryogenesis, and carcinogenesis. Inheritance of the epigenetic methylation pattern is mediated by the enzyme DNA methyltransferase 1 (Dnmt1), which methylates newly synthesized CpG sequences during DNA replication, depending on the methylation status of the template strands. The protein UHRF1 (also known as Np95 and ICBP90) recognizes hemi-methylation sites via a SET and RING-associated (SRA) domain and directs Dnmt1 to these sites. Here we report the crystal structures of the SRA domain in free and hemi-methylated DNA-bound states. The SRA domain folds into a globular structure with a basic concave surface formed by highly conserved residues. Binding of DNA to the concave surface causes a loop and an amino-terminal tail of the SRA domain to fold into DNA interfaces at the major and minor grooves of the methylation site. In contrast to fully methylated CpG sites recognized by the methyl-CpG-binding domain, the methylcytosine base at the hemi-methylated site is flipped out of the DNA helix in the SRA-DNA complex and fits tightly into a protein pocket on the concave surface. The complex structure suggests that the successive flip out of the pre-existing methylated cytosine and the target cytosine to be methylated is associated with the coordinated transfer of the hemi-methylated CpG site from UHRF1 to Dnmt1.     

Structural basis of actin filament nucleation and processive capping by a formin homology 2 domain

The conserved formin homology 2 (FH2) domain nucleates actin filaments and remains bound to the barbed end of the growing filament. Here we report the crystal structure of the yeast Bni1p FH2 domain in complex with tetramethylrhodamine-actin. Each of the two structural units in the FH2 dimer binds two actins in an orientation similar to that in an actin filament, suggesting that this structure could function as a filament nucleus. Biochemical properties of heterodimeric FH2 mutants suggest that the wild-type protein equilibrates between two bound states at the barbed end: one permitting monomer binding and the other permitting monomer dissociation. Interconversion between these states allows processive barbed-end polymerization and depolymerization in the presence of bound FH2 domain. Kinetic and/or thermodynamic differences in the conformational and binding equilibria can explain the variable activity of different FH2 domains as well as the effects of the actin-binding protein profilin on FH2 function.     

Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation

Both prokaryotic and eukaryotic organisms contain DNA bridging proteins, which can have regulatory or architectural functions. The molecular and mechanical details of such proteins are hard to obtain, in particular if they involve non-specific interactions. The bacterial nucleoid consists of hundreds of DNA loops, shaped in part by non-specific DNA bridging proteins such as histone-like nucleoid structuring protein (H-NS), leucine-responsive regulatory protein (Lrp) and SMC (structural maintenance of chromosomes) proteins. We have developed an optical tweezers instrument that can independently handle two DNA molecules, which allows the systematic investigation of protein-mediated DNA-DNA interactions. Here we use this technique to investigate the abundant non-specific nucleoid-associated protein H-NS, and show that H-NS is dynamically organized between two DNA molecules in register with their helical pitch. Our optical tweezers also allow us to carry out dynamic force spectroscopy on non-specific DNA binding proteins and thereby to determine an energy landscape for the H-NS-DNA interaction. Our results explain how the bacterial nucleoid can be effectively compacted and organized, but be dynamic in nature and accessible to DNA-tracking motor enzymes. Finally, our experimental approach is widely applicable to other DNA bridging proteins, as well as to complex DNA interactions involving multiple DNA molecules.     

Conformational dynamics of individual DNA molecules during gel electrophoresis

Gel electrophoresis is widely used in molecular biology to separate DNA molecules according to their sizes. The physical basis of this size separation is, however, poorly understood. Here we report observations of individual, fluorescently stained DNA molecules as they migrate during various kinds of gel electrophoresis. Their movement, under the influence of either a steady electric field or a pulsed-field, is characterized by cycles of elongation and contraction. Initially relaxed coils of DNA lengthen into 'hook-shaped' configurations which temporarily 'hang-up' on obstacles in the gel matrix before sliding off, contracting and entering another cycle. The effects of a new electrophoresis technique, termed 'pulse-oriented electrophoresis', which allows the effective angle of the electric field, and hence the molecular orientation of DNA, to be varied without electrode rearrangement, are also studied. In this case the DNA adopts a 'staircase' configuration showing that the net orientation in a direction is given by the vector sum of the pulses used.     

光谱法研究卟啉-蒽醌化合物及其金属锌配合物与DNA的相互作用

用光谱法研究以二肽链连接的卟啉-蒽醌化合物及其金属锌配合物与DNA的相互作用.结果表明:卟啉-蒽醌化合物及其金属锌配合物与DNA发生外部结合.由紫外-可见光谱滴定数据进行拟合计算出卟啉-蒽醌化合物及其金属锌配合物与DNA相互作用的结合常数分别为2.2×105(mol/L)-1和6.7×105(mol/L)-1.     

Expression of functional human interleukin-2 receptor in mouse T cells by cDNA transfection

Interleukin-2 (IL-2) in combination with the IL-2 receptor has an essential role in antigen-stimulated proliferation of T lymphocytes. It has been proposed that the constitutive expression of the IL-2 receptor on adult T-cell leukaemia (ATL) cells may be associated with transformation of T cells. Although we and others have isolated complementary DNA clones encoding a protein that binds IL-2, formal proof that this protein is the IL-2 receptor requires demonstration of IL-2-dependent growth stimulation of cells expressing the protein. In addition, a functional assay system other than binding of IL-2 is required to investigate the molecular mechanism of signal transmission through the IL-2 receptor using artificially mutated cDNA. The IL-2 receptor expressed in non-lymphoid cells by cDNA transfection did not mediate a growth signal, implying that lymphoid cells expressing the functional receptor might have specific accessory molecule(s) for signal transmission by the receptor. Therefore, we established a line of IL-2-dependent mouse cells (CT/hR) expressing both murine (endogenous) and human IL-2 receptors. Here, by blocking the endogenous mouse IL-2 receptors with monoclonal antibodies, we show that the human IL-2 receptor of CT/hR cells is functionally active. Although CT/hR expressed the human IL-2 receptor constitutively, growth of these cells was strictly dependent on IL-2, indicating that uncontrolled over-expression of the IL-2 receptor was not by itself sufficient for T-cell transformation.     

Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA

Two crystal structures of the glucocorticoid receptor DNA-binding domain complexed with DNA are reported. The domain has a globular fold which contains two Zn-nucleated substructures of distinct conformation and function. When it binds DNA, the domain dimerizes, placing the subunits in adjacent major grooves. In one complex, the DNA has the symmetrical consensus target sequence; in the second, the central spacing between the target's half-sites is larger by one base pair. This results in one subunit interacting specifically with the consensus target half-site and the other nonspecifically with a noncognate element. The DNA-induced dimer fixes the separation of the subunits' recognition surfaces so that the spacing between the half-sites becomes a critical feature of the target sequence's identity.     

染色体结构维持蛋白Smc5/6复合体的结构与功能

真核生物基因组DNA主要以染色体的形式存在于细胞核中，染色体结构的稳定及其动态变化对于真核生物遗传信息从亲代到子代中的准确传递和维持细胞的正常功能是必不可少的。染色体结构维持蛋白（Structure Maintenance of Chromosome，Smc）在染色体结构维持及DNA损伤修复方面发挥着关键性的作用。Smc蛋白家族包括3类：黏连蛋白（Cohesin）、凝缩蛋白（Condesin）和Smc5/6复合体（Smc5/6 complex）。Smc5/6复合体在真核生物中分布广泛且高度保守，在DNA损伤同源重组修复、DNA复制、端粒长度维持及胚胎发育中发挥重要作用。本文系统介绍了Smc5/6复合体结构特征和生物学功能领域的相关进展，为深入研究Smc5/6复合体提供理论参考。     

Phosphorylation-dependent binding of mitotic cyclins to Cdc6 contributes to DNA replication control

Cyclin-dependent kinases (CDKs) limit the activation of DNA replication origins to once per cell cycle by preventing the assembly of pre-replicative complexes (pre-RCs) during S, G2 and M phases of the cell cycle in the budding yeast Saccharomyces cerevisiae. CDKs inhibit each pre-RC component (ORC, Cdc6, Cdt1/Mcm2-7) by different mechanisms. We show here that the mitotic CDK, Clb2/Cdc28, binds tightly to an amino-terminal domain (NTD) of Cdc6, and that Cdc6 in this complex is unable to assemble pre-RCs. We present evidence indicating that this Clb2-dependent mechanism contributes to preventing re-replication in vivo. CDK interaction with the NTD of Cdc6 is mediated by the cyclin subunit Clb2, and could be reconstituted with recombinant Clb2 protein and synthetic NTD peptides. Tight Clb2 binding occurred only when the NTD was phosphorylated on CDK consensus sites. Human CDKs containing cyclins A, B and E also bound specifically to phospho-NTD peptides. We propose that direct binding of cyclins to phosphopeptide motifs may be a widespread phenomenon contributing to the targeting of CDKs to substrates.     

Nir2碳端结构域的晶体结构

Nir2蛋白具有多个结构域，在生物体中可能发挥多种生理功能。本实验用MAD软件对大肠杆菌BL21（plys）原核表达的Nir2蛋白碳端进行了晶体结构分析。结果显示，硒蛋氨酸取代蛋白晶体及未取代蛋白晶体同属空间群P212121,晶胞参数a=109.468 Å, b=120.621 Å, c=70.315 Å，分辨率分别为2.2 Å和2.7 Å。一个不对称单位含有三个分子，含水量为48%。Nir2蛋白碳端由C片层和N片层构成，其中C片层与钙ATPase的催化区域P相似，N片层为许多不相关的功能性蛋白所共有。研究证明二者之间的静电作用是影响Nir2蛋白碳端与Pyk2 FERM区域结合的主要因素。该晶体结构的分析结果为进一步研究Nir2蛋白碳端的潜在功能提供了结构基础。     

甲基丙烯酸-8-羟基喹啉-Eu(Ⅲ)配合物与鲱鱼精DNA的作用机制

采用光谱法、黏度法和DNA热变性方法研究甲基丙烯酸8羟基喹啉Eu(Ⅲ)配台物(Eu(MA)_2(hq))与鲱鱼精DNA之间的作用机制和结台常数结果表明:该配台物加人鲱鱼精DNA后,特征吸收峰发生明显的减色效应,但峰位红移现象不明显;该配台物能猝灭中性红DNA体系的荧光;该配台物与鲱鱼精DNA的结台常数K_(20)=5.91×10~3L/mol,K_(35)=7.70×10~3L/mol,二者作用的物质的量比为1;该配台物与鲱鱼精DNA之间的热力学函数△rH_m~e=1.34×10~3J/mol,△rG_m~e=-2.12×10~4J/mol,△rS_m~e=76.41 J/(mol·K);鲱鱼精DNA的相对黏度增大,熔点明显升高,Eu(MA)_2(hq)与鲱鱼精DNA之间的作用模式为插人作用.