Mechanism of Microbial DNA Hypervariation through Mutagenic Transposition

项目摘要 多样性产生逆转录元件(DGR)是在细菌中发现的分子进化机器， 古生菌及其病毒。它们使蛋白质编码序列多样化，以促进 它们的宿主适应不断变化的环境。超变异是由容易出错的逆转位引起的 这个过程称为突变定位，它从模板重复(Tr)中转移序列信息 到可变重复(VR)，从而导致腺嘌呤到随机核苷酸的转换。长期目标 目的是了解DGR突变归巢的机制，并将其发展为 实际应用。类似于相关的逆转录元件，建议发生DGR归巢 通过靶DNA启动的逆转录机制。有趣的是，最近的发现 Pi的研究小组发现，波尔德氏菌噬菌体DGR的逆转录是由一种 下游的腺嘌呤残基是RNA的中间体，是靶标(VR)不依赖的。有趣的是， 在依赖于BRT(波氏杆菌逆转录酶)的BRT中发现TRRNA中间体被缺口 在RT催化下产生3‘-OH的方式用于cDNA的启动和单一氨基酸的取代 CORE取消了划痕活性，表明BRT在切割反应中起催化作用。 腺嘌呤特异性突变发生在(-)cDNA合成过程中，其结果是错误结合 BRT法测定标准脱氧核糖核苷酸。此外，突变分析显示，这一特殊的， 靶标非依赖性逆转录反应负责DGR突变归巢，揭示 DNA超变的一种新机制。具体的目标是基于这些新发现。 目标1将表征TRRNA的二级结构，并将Avd和BRT结合位点映射到 核糖核酸中间体。这些研究将产生DGR RNA的第一个二级结构，并且 揭示TRRNA中间体是否被BRT催化切割。目标2将决定 Vr基因3‘端和5’端的基因整合机制碱基配对相互作用的作用 在3‘cDNA整合中，RNA引物与VR DNA之间以及cDNA与VR DNA之间的相互作用 测试过。这些研究可能导致发现新的cdna整合机制。目标3将 确定腺嘌呤特异性突变的机制，这是DGRS的一个标志。 了解腺嘌呤特异性突变的机制可能会对RT产生新的见解 忠诚度问题。总之，本申请中提出的研究将阐明DGR的机制 突变归巢，这可能在健康和科学方面有广泛的影响。

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.