Mechanism of Microbial DNA Hypervariation through Mutagenic Transposition

通过诱变转座导致微生物 DNA 高度变异的机制

基本信息

批准号：
9788497
负责人：
Donald H Burke
金额：
$ 28.88万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-19 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9788497
关键词：
Address Adenine Amino Acid Substitution Archaea Bacteria Bacteriophages Base Pairing Binding Sites Bioinformatics Biomedical Engineering Bordetella Catalytic Domain Cleaved cell Complementary DNA Complex DNA DNA Sequence Detection Development Elements Environment Family Genes Genetic Genetic Polymorphism Goals Health Sciences Homing Hybrids Integration Host Factors Investigation Lead Light Maps Mediating Metagenomics Methodology Molecular Evolution Mutagenesis Mutation Mutation Analysis Nucleotides Paper Pathogenesis Play Process Property Proteins RNA RNA Sequences RNA primers RNA-Directed DNA Polymerase Reaction Research Retroelements Retrotransposition Reverse Transcription Ribonucleoproteins Role Site Structure Testing Tropism Virus Work base in vivo insight microbial novel nucleic acid binding protein practical application receptor

项目摘要

Project Abstract Diversity-generating retroelements (DGRs) are molecular evolution machines found in bacteria, archaea and their viruses. They diversify protein-encoding sequences to facilitate the adaptation of their hosts to changing environments. Hypervariation results from an error-prone retrotransposition process called mutagenic homing, which transfers sequence information from a template repeat (TR) to a variable repeat (VR) that results in adenine to random nucleotide conversions. The long-term goal of the PI’s group is to understand the mechanism of DGR mutagenic homing and to develop them for practical applications. In analogy to related retroelements, DGR homing was proposed to occur through a target DNA-primed reverse transcription mechanism. Interestingly, recent discoveries by the PI’s group showed that reverse transcription of TR of the Bordetella phage DGR is primed by a downstream adenine residue of the RNA intermediate and is target (VR)-independent. Intriguingly, the TR RNA intermediate was found to be nicked in a bRT (Bordetella reverse transcriptase)-dependent manner to generate a 3’-OH for cDNA priming, and single amino acid substitutions at the RT catalytic core abolish the nicking activity, suggesting that bRT plays a catalytic role in the cleavage reaction. Adenine-specific mutagenesis occurs during (–)cDNA synthesis and results from misincorporation of standard deoxyribonulceotides by bRT. In addition, mutational analysis showed that this special, target-independent reverse transcription reaction is responsible for DGR mutagenic homing, revealing a novel mechanism of DNA hypervariation. The specific aims are based on these novel discoveries. Aim 1 will characterize the secondary structure of TR RNA and map the Avd and bRT binding sites on the RNA intermediate. These studies will generate the first secondary structure of a DGR RNA, and reveal whether the TR RNA intermediate is catalytically cleaved by bRT. Aim 2 will determine the mechanism of cDNA integration at the 3’ and 5’ ends of VR. Roles of base pairing interactions between the RNA primer and VR DNA and between cDNA and VR DNA in 3’ cDNA integration will be tested. These studies may lead to discovery of novel cDNA integration mechanisms. Aim 3 will determine the mechanism of adenine-specific mutagenesis, which is a hallmark of DGRs. Understanding the mechanism of adenine-specific mutagenesis will likely yield new insights on RT fidelity issues. In summary, studies proposed in this application will elucidate the mechanism of DGR mutagenic homing, which may have broad implications in health and science.

项目摘要产生多样性的恢复元素（DGRS）是细菌中发现的分子进化机，古细菌及其病毒。它们使蛋白质编码序列多样化，以促进适应他们的主机进入不断变化的环境。过变量是由易逆转录转词引起的工艺称为诱变归巢，从模板重复转移序列信息（TR）可变重复（VR），导致腺嘌呤到随机核苷酸转化率。长期目标 PI的小组是了解DGR诱变归宿的机制，并为其开发实际应用。类似于相关的追溯元素，提出了DGR归巢通过靶DNA键入的逆转录机制。有趣的是，最近发现 Pi的组表明，Bordetella噬菌体DGR的TR逆转录由A启动 RNA中间体的下游腺嘌呤居住区，是靶（VR）独立的。有趣的是，发现TR RNA中间体被划入BRT（Bordetella逆转录酶）依赖性生成用于cDNA启动的3'-OH的方式，以及在RT催化的单个氨基酸取代核心废除了划痕活性，表明BRT在切割反应中起催化作用。腺嘌呤特异性诱变发生在（ - ）cDNA合成期间，并导致因掺杂而导致 BRT的标准Deoxyribonulceotides。此外，突变分析表明，这个特殊的目标无关的逆转录反应是DGR诱变的归因，揭示了 DNA过变量的新型机制。具体目的是基于这些新颖的发现。 AIM 1将表征TR RNA的二级结构，并绘制AVD和BRT结合位点 RNA中间体。这些研究将产生DGR RNA的第一个二级结构，并且揭示TR RNA中间体是否被BRT催化。 AIM 2将确定 VR的3'和5'末端的cDNA整合机理。基础配对相互作用的角色在RNA底漆和VR DNA之间以及3’cDNA积分中的cDNA和VR DNA之间将是测试。这些研究可能导致发现新型cDNA整合机制。目标3意志确定腺嘌呤特异性诱变的机理，这是DGRS的标志。了解腺嘌呤特异性诱变的机制可能会对RT产生新的见解忠诚问题。总而言之，此应用中提出的研究将阐明DGR的机制诱变的归巢，可能对健康和科学具有广泛的影响。