Knowledge Resource Center for Ecological Environment in Arid Area
| 项目编号 | 18-74-00049 |
| Nicking endonucleases: biological role in bacteria of Bacillus genus, new possibilities of application in molecular biology and biotechnology | |
| Abrosimova Liudmila | |
| 主持机构 | Federal State Budgetary Educational Institution of Higher Education Lomonosov Moscow State University, |
| 开始日期 | 2018 |
| 结束日期 | 2019 |
| 资助机构 | RU-RSF（俄罗斯科学基金会） |
| 语种 | 英语 |
| 国家 | 俄罗斯 |
| 中文简介 | 04 - BIOLOGY AND LIFE SCIENCES, 04-208 - Molecular biology |
| 英文简介 | The fundamental scientific goal of this project is studying the biological role of nicking endonucleases (NE). They recognize short sequences in double-stranded DNA and cleave only one strand. According to restriction endonuclease database REBASE almost all known natural NEs are purified from the genus Bacillus. This fact allows to suggest that formation the heterodimeric restriction endonucleases (REs) – the components of restriction-modification system, is not the only role of NEs. They can also participate in the processes involving introduction of single-strand breaks such as recombination and DNA repair. Determining the role of these enzymes in functioning of the cell will clarify a matter of adaptation of Bacillus bacteria to changing environmental factors, allow to control their survival in different conditions and extend the field of their practical application. The major part of bacteria from Bacillus genus is not characterized to the full extent. The object of the study is Bacillus species D6 strain from Sahara Desert. In previous studies NE BspD6I was purified from this strain and its properties were characterized. NE BspD6I recognizes 5'-GAGTC-3'/5'-GACTC-3' sequence that is often present in promotors of the phage genes, such as T7 bacteriophage. NE BspD6I hydrolyzes the “top” DNA strand after 4th nucleotide from the recognition site in 3'-direction. We suggest to use NE BspD6I as an instrument for studying the functionally related MutL proteins with endonuclease activity that are important and poorly studied components of mismatch repair system (MMR). We also propose the approaches for wider practical usage of NE. To solve the indicated problems we plan to develop several directions. We will quantitatively assess the NE expression level in Bacillus species D6 by means of Western blot method. The genome of Bacillus species D6 will be sequenced and annotated. The comparison of accessible genomes of Bacillus halodurans C-125 and Geobacillus stearothermophilus with the one of Bacillus species D6 will allow to determine their conservative and unique properties. The obtained results will give the opportunity to establish the evolutionary interrelation for bacteria which possess isoschizomeric NE. The presence or absence of sequences coding MMR proteins or involved in homologous recombination in Bacillus species D6 genome can clarify the issue about NE BspD6I cellular functions that differ from participation in the restriction-modification process. We plan to experimentally determine the role of NE BspD6I in life cycle of Bacillus sp. D6. For this purpose the phenotypic analysis of wild type cells and cells with knockout of the NE BspD6I gene will be performed. For control experiments the complementary strain containing the deleted NE BspD6I gene will be constructed. We plan to study the cell morphology of wild type and mutant strains, analyze the growing peculiarities of the cells in different conditions. Investigating the biological role of NE BspD6I can lead to discovering the new mechanisms of DNA repair and can help to better understand the functioning of socially important bacteria from Bacillus genus. For the first time we suggest to use NE BspD6I as an instrument for fundamental investigations namely for studying the functioning of MutL protein from Neisseria gonorrhoeae (NgoL) that is a human pathogen. We will produce the chimeric proteins combining domains of NE BspD6I, NgoL and firm or flexible linker between them, investigate their ability to hydrolyze DNA and determine the position of hydrolysis. Studying of NgoL functions will allow to broaden the fundamental knowledge about properties of rare enzymes hydrolyzing only one DNA strand. This information can be also helpful for future design of medicine inhibiting the functioning of Neisseria gonorrhoeae. The described original chimeric constructs can also serve as a basis for design of the enzymes with new specificity and hydrolysis of unique DNA sequences that is necessary in diagnostic applications and genome editing. The ability of NEs to function as independent enzymes caused their essential role in molecular biology and biotechnology. We suggest the ways for wider practical use of these enzymes. Particularly we plan to test the original method for temporal blocking of NE BspD6I activity that can substantially increase the efficacy of isothermal DNA amplification reaction. The approach is based on usage of non-hydrolyzable substrate analogue of NE BspD6I (the “trap”) that can form nonproductive complex with the enzyme. The transient “switching on” the enzymatic activity at higher temperatures due to the dissociation of the “trap” from the complex with NE should essentially minimize the quantity of by-products which are formed after additional action of NE during DNA amplification. All the tasks related to determination of the NE BspD6I biological role in the cell and developing new approaches for NE application are presented here for the first time on the ground of literature analysis and preliminary data obtained by the leader of the project. |
| 英文关键词 | nicking endonucleases DNA-protein interaction DNA mismatch repair system |
| 来源学科分类 | 04 - BIOLOGY AND LIFE SCIENCES, 04-208 - Molecular biology |
| 资源类型 | 项目 |
| 条目标识符 | http://119.78.100.177/qdio/handle/2XILL650/356053 |
| 推荐引用方式 GB/T 7714 | Abrosimova Liudmila.Nicking endonucleases: biological role in bacteria of Bacillus genus, new possibilities of application in molecular biology and biotechnology.2018. |
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