解开生命之谜的罗塞达石碑 ——纪念沃森、克里克发现DNA双螺旋结构50周年

1953年2月28日,在英国剑桥的伊尔(Eagle)酒馆,37岁的英国科学家克里克向在场吃午饭的人(主要是一些科学家)宣布,他和25岁的美国同事沃森已经完成了一项伟大的科学发现——建立了DNA双螺旋结构分子模型,从而揭示了生命的奥秘。消息传出,满座皆惊。不久,他们的论文《核酸的分子结构——脱氧核糖核酸的结构》和《脱氧核糖核酸结构的遗传学意义》两篇文章相继于4月25日和5月30日在英国权威科学杂志《自然》上刊出。     

DNA双螺旋结构发现背后的女性——纪念罗莎琳德·富兰克林逝世49周年

罗莎琳德·富兰克林,一位擅长晶体学研究的科学家,她的DNA晶体X射线衍射照片对DNA结构的发现起到了关键作用.但是,这位37岁英年早逝,与诺贝尔奖擦身而过的伟大女性却一直备受争议.简介了罗莎琳德·富兰克林博士短暂的一生,并论述她在DNA双螺旋结构发现中的杰出贡献.     

中国科学院古脊椎动物与古人类研究所成立暨北京猿人第一个头盖骨发现65周年庆典在京举行

中国科学院古脊椎动物与古人类研究所成立暨北京猿人第一个头盖骨发现６５周年庆典在京举行１９９４年是北京猿人第一个头盖骨发现６５周年,也是古脊椎动物与古人类所的前身──中国地质调查所新生代研究室成立６５周年;更重要的是古脊椎所几代人盼望已久并为之做出不懈...     

Rad50 Zinc Hook Is Important for the Mre11 Complex to Bind Chromosomal DNA Double-stranded Breaks and Initiate Various DNA Damage Responses

The Mre11-Rad50-Nbs1 (MRN) complex plays critical roles in checkpoint activation and double-stranded break (DSB) repair. The Rad50 zinc hook domain mediates zinc-dependent intercomplex associations of MRN, which is important for DNA tethering. Studies in yeast suggest that the Rad50 zinc hook domain is essential for MRN functions, but its role in mammalian cells is not clear. We demonstrated that the human Rad50 hook mutants are severely defective in various DNA damage responses including ATM (Ataxia telangiectasia mutated) activation, homologous recombination, sensitivity to IR, and activation of the ATR pathway. By using live cell imaging, we observed that the Rad50 hook mutants fail to be recruited to chromosomal DSBs, suggesting a novel mechanism underlying the severe defects observed for the Rad50 hook mutants. In vitro analysis showed that Zn(2+) promotes wild type but not the hook mutant of MR to bind double-stranded DNA. In vivo, the Rad50 hook mutants are defective in being recruited to chromosomal DSBs in both H2AX-proficient and -deficient cells, suggesting that the Rad50 hook mutants are impaired in direct binding to chromosomal DSB ends. We propose that the Rad50 zinc hook domain is important for the initial binding of MRN to DSBs, leading to ATM activation to phosphorylate H2AX, which recruits more MRN to the DSB-flanking chromosomal regions. Our studies reveal a critical role for the Rad50 zinc hook domain in establishing and maintaining MRN recruitment to chromosomal DSBs and suggest an important mechanism of how the Rad50 zinc hook domain contributes to DNA repair and checkpoint activation.     

Structure of the Rad50 DNA double‐strand break repair protein in complex with DNA

The Mre11–Rad50 nuclease–ATPase is an evolutionarily conserved multifunctional DNA double‐strand break (DSB) repair factor. Mre11–Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50^NBD (nucleotide‐binding domain) in complex with Mre11^HLH (helix‐loop‐helix domain), AMPPNP, and double‐stranded DNA. DNA binds between both coiled‐coil domains of the Rad50 dimer with main interactions to a strand‐loop‐helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double‐strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP‐bound state.     

Direct Evidence for the Formation of Precatenanes during DNA Replication

The dynamics of DNA topology during replication are still poorly understood. Bacterial plasmids are negatively supercoiled. This underwinding facilitates strand separation of the DNA duplex during replication. Leading the replisome, a DNA helicase separates the parental strands that are to be used as templates. This strand separation causes overwinding of the duplex ahead. If this overwinding persists, it would eventually impede fork progression. In bacteria, DNA gyrase and topoisomerase IV act ahead of the fork to keep DNA underwound. However, the processivity of the DNA helicase might overcome DNA gyrase and topoisomerase IV. It was proposed that the overwinding that builds up ahead of the fork could force it to swivel and diffuse this positive supercoiling behind the fork where topoisomerase IV would also act to maintain replicating the DNA underwound. Putative intertwining of sister duplexes in the replicated region are called precatenanes. Fork swiveling and the formation of precatenanes, however, are still questioned. Here, we used classical genetics and high resolution two-dimensional agarose gel electrophoresis to examine the torsional tension of replication intermediates of three bacterial plasmids with the fork stalled at different sites before termination. The results obtained indicated that precatenanes do form as replication progresses before termination.     

Purification and cloning of DNA fragments fractionated on agarose gels

Purification of DNA fragments from acrylamide or agarose gels is a commonly used technique in the molecular biology laboratory. This article describes a rapid, efficient, and inexpensive method of purifying DNA fractions from an agarose gel. The purified DNA is suitable for use in a wide range of applications including ligation using DNA ligase. The procedure uses standard high-melting-temperature agarose and normal TBE electrophoresis buffer. In addition, the protocol does not involve the use of highly toxic organic solvents such as phenol.     

利用同序异尾限制性内切酶快速克隆多基因片段的新方法

介绍一种可以快速进行多基因片段克隆的新方法——同序异尾限制性内切酶(isoschizomer-heterotailrestrictionendonuclease,IHRE)一步克隆法,该方法可以一步完成多达6个DNA片段的连接,具有简单快速、节省试剂、成功率高、不引入非目的基因序列等很多优点.应用该方法已经成功完成对人肠激酶轻链多基因克隆、HSV多表位DNA疫苗的构建等.     

Structural mechanism of endonucleolytic processing of blocked DNA ends and hairpins by Mre11-Rad50

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Pharmacophore modeling and molecular dynamics approach to identify putative DNA Gyrase B inhibitors for resistant tuberculosis

One of the major mechanisms followed by the therapeutic agents to target the causative organism of TB, mycobacterium tuberculosis (Mtb), involves disruption of the replication cycle of the pathogen DNA. The process involves two steps that occur simultaneously, ie, breakage and reunion of DNA at gyrase A (GyrA) domain and ATP hydrolysis at gyrase B (GyrB) domain. Current therapy for multi-drug resistant TB involves FDA approved, Fluoroquinolone-based antibiotics, which act by targeting the replication process at GyrA domain. However, resistance against fluoroquinolones due to mutations in the GyrA domain has limited the use of this therapy and shifted the focus of the research community on the GyrB domain. Thus, this study involves in silico designing of chemotherapeutic agents for resistant TB by targeting GyrB domain. In the current study, a pharmacophore model for GyrB domain was generated using reported inhibitors. It was utilized as a query search against three commercial databases to identify GyrB domain inhibitors. Additionally, a qualitative Hip-Hop pharmacophore model for GyrA was also developed on the basis of some marketed fluoroquinolone-based GyrA inhibitors, to remove non-selective gyrase inhibitors obtained in virtual screening. Further, molecular dynamic simulations were carried out to determine the stability of the obtained molecules in complex with both the domains. Finally, Molecular mechanics with generalized Born and surface area solvation score was calculated to determine the binding affinity of obtained molecule with both domains to determine the selectivity of the obtained molecules that resulted in seven putative specific inhibitors of GyrB domain.