Structure and function of novel prokaryotic DNA transposases

新型原核DNA转座酶的结构和功能

• 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
• 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

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 转座酶家族的代表性成员进行的体外生化和结构联合研究表明，该家族使用一种全新的重组途径，仅涉及单链 DNA 的运动。一个特别令人惊讶的发现是，转座酶通过 DNA-DNA 相互作用而不是使用位点特异性 DNA 结合域来识别其靶位点：靶位点识别是通过靶位点与转座子 DNA 内部片段之间的碱基配对相互作用来完成的。这表明通过改变内部片段，可以将目标定向到新的目标位点。如果我们能做到这一点，就可以将外源基因精确引入染色体的良性位置或可以以细胞和发育特异性方式适当控制基因表达的位置。 在我们最近的工作中，我们一直在继续探索IS200/IS605转座的机制。特别是，我们一直在研究转座子末端和识别 DNA 发夹之间的核苷酸数量（“接头长度”）如何影响 IS608 转座，以及拟议的结构变化如何驱动从 DNA 链切割到链转移的过程。我们的数据与我们之前提出的旋转模型一致，其中两个灵活的α螺旋相对于酶活性位点交替其构型，并且这些构型之间的来回 - 以及“重置”步骤 - 驱动转座反应前进。 我们还一直在研究与细菌重复外源回文序列（或 REP）相关的推定转座酶。 REP 形成核苷酸茎环结构，并且被发现大量分散在许多细菌物种中。它们的庞大数量表明存在一个导致它们在宿主物种中扩张的过程，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，我们确定了大肠杆菌菌株 MG1655 中 TnpA(REP) 与 DNA 回文体复合物的结构。事实上，它类似于 IS200/IS605 转座酶，并且具有能够切割某些包含 REP 序列的 DNA 结构的特性。因此，它似乎负责 REP 序列在整个细菌基因组中的增殖，并且是基因组进化的重要贡献者。 Curcio, M.J. 和德比郡, K.M. (2003) 国家。莫尔牧师。细胞。生物。 4、865-877。 Debets-Ossenkopp，Y.J.，等人。 (1999) 抗菌剂。特工化疗。 43、2657-2662。 Kersulyte，D.，等人。 (2002)细菌杂志。 184、992-1002。 Mennecier, S.、Servant, P.、Coste, G.、Bailone, A. 和 Sommer, S. (2006) Mol。微生物。 59、317-325。 Sebaihia，M.等人。 （2006）自然基因。 38、779-786。