Structure and function of novel prokaryotic DNA transposases

新型原核DNA转座酶的结构和功能

基本信息

批准号：
8741429
负责人：
Frederick Dyda
金额：
$ 40.52万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8741429
关键词：
Active Sites Affect Amino Acids Antibiotic Resistance Antibiotics Back Bacteria Bacterial Antibiotic Resistance Bacterial Genome Base Pairing Benign Biochemical Cells Characteristics Chromosomes Cleaved cell Clostridium difficile Combined Modality Therapy Complex DNA DNA Binding Domain DNA Insertion Elements DNA Structure DNA Transposons Data Development Elements Environment Enzymes Escherichia coli Event Evolution Family Gastritis Gene Expression Genes Genetic Recombination Genets Genome Goals Health Helicobacter pylori Hospitals In Vitro Intervention Inverted Terminal Repeat Lead Length Location Mediating Metronidazole Modeling Movement Nature Nitroreductases Nucleotides Pathology Pathway interactions Population Process Property Proteins Reaction Relative (related person)Research Resistance Rotation Single-Stranded DNA Site Structure System Transposase Ulcer Virulent Work alpha helix flexibility gene therapy malignant stomach neoplasm member novel nuclease programs protein folding stem structural biology

项目摘要

Our combined in vitro biochemical and structural studies on a representative member of the IS200/IS605 transposase family demonstrated that this family uses a completely novel recombination pathway involving the movement of only single-stranded DNA. One particularly surprising discovery was that the transposase recognizes its target site through DNA-DNA interactions rather than using a site-specific DNA binding domain: target site recognition is accomplished by base pairing interactions between the target site and an internal segment of transposon DNA. This suggests the possibility that by changing the internal segment, targeting could be directed to novel target sites. If we can do this, this might allow the precise introduction of exogenous genes into benign locations in chromosomes or places where gene expression can be appropriately controlled in a cell- and development-specific manner. In our recent work, we have been continuing to explore the mechanism of IS200/IS605 transposition. In particular, we have been investigating how the number of nucleotides between the transposon ends and the recognition DNA hairpin (the "linker length") affects IS608 transposition, and also how a proposed structural change drives the process from DNA strand cleavage to strand transfer. Our data is consistent with our previously proposed rotation model in which two flexible alpha-helices alternate their configuration with respect to the enzyme active sites, and that the back-and-forth between these configurations - along with a "reset" step - drives the transposition reaction forward. We have also been studying the putative transposase associated with bacterial Repeated Extragenic Palindromic Sequences (or REPs). REPs form nucleotide stem-loop structures and are found scattered in high numbers in many bacterial species. Their sheer number suggests there was a process that led to their expansion in their host species, and it has been proposed that this might involve an protein closely related to the IS200/IS605 transposases. To confirm this, we determined the structure of the TnpA(REP) from E. coli strain MG1655 in complex with a DNA palindrome. Indeed, it resembles the IS200/IS605 transposases and shares the property of being able to cleave certain DNA structures that contain REP sequences. Thus, it appears likely that it has been responsible for the proliferation of REP sequences throughout bacterial genomes, and has been an important contributor to genome evolution. Curcio, M.J. and Derbyshire, K.M. (2003) Nat. Rev. Mol. Cell. Biol. 4, 865-877. Debets-Ossenkopp, Y.J., et al. (1999) Antimicrob. Agents Chemother. 43, 2657-2662. Kersulyte, D., et al. (2002) J. Bacteriol. 184, 992-1002. Mennecier, S., Servant, P., Coste, G., Bailone, A., and Sommer, S. (2006) Mol. Microbiol. 59, 317-325. Sebaihia, M. et al. (2006) Nature Genet. 38, 779-786.

我们对IS200/IS605转座酶家族代表性成员的体外生化和结构研究的组合表明，该家族使用了一种完全新颖的重组途径，涉及仅单链DNA的运动。一个特别令人惊讶的发现是，转座酶通过DNA-DNA相互作用识别其目标位点，而不是使用位点特异性的DNA结合域：通过碱基对位点与转座子DNA的内部段之间的基础配对相互作用来实现目标位点识别。这表明，通过更改内部段，靶向可以将目标定向到新的目标位点。如果我们可以做到这一点，这可能会允许将外源基因精确地引入染色体或可以以细胞和发育特异性方式适当控制基因表达的地方的良性位置。在我们最近的工作中，我们一直在继续探索IS200/IS605换位的机制。特别是，我们一直在研究转座子末端和识别DNA发夹（“接头长度”）之间的核苷酸的数量如何影响IS608换位，以及提出的结构变化如何将过程从DNA Strand裂解转移到链转移。我们的数据与我们先前提出的旋转模型一致，在该模型中，两个柔性α-螺旋可与酶的活性位点交替使用它们的配置，并且这些配置之间的来回传动 - 加上“重置”步骤 - 驱动转置反应。我们还一直在研究与细菌重复的外胞外粒细胞序列（或reps）相关的推定转座酶。代表形成核苷酸干循环结构，发现在许多细菌物种中散布着高数量。他们的数字表明，有一个过程导致它们在宿主物种中的扩张，并且有人提出这可能涉及与IS200/IS605转座酶密切相关的蛋白质。为了证实这一点，我们确定了与DNA palindrome复合物中大肠杆菌菌株Mg1655的TNPA（REP）的结构。实际上，它类似于IS200/IS605转座酶，并具有能够裂解包含REP序列的某些DNA结构的属性。因此，它似乎已经导致了整个细菌基因组的REP序列的增殖，并且是基因组进化的重要贡献。 M.J. Curcio和K.M. Derbyshire （2003）Nat。摩尔牧师。细胞。生物。 4，865-877。 DEBETS-OSSENKOPP，Y.J。等。（1999）Antimicrob。代理化学者。 43，2657-2662。 Kersulyte，D。等。（2002）J。Bacteriol。 184，992-1002。 Mennecier，S.，Servant，P.，Coste，G.，Bailone，A。和Sommer，S。（2006）Mol。微生物。 59，317-325。 Sebaihia，M。等。（2006）自然基因。 38，779-786。