Mechanism of Microbial DNA Hypervariation through Mutagenic Transposition

项目摘要 多样性生成逆转录因子（DGR）是在细菌中发现的分子进化机器， 古细菌和它们的病毒它们使蛋白质编码序列多样化，以促进 适应不断变化的环境高变异是由一个容易出错的逆转录转座引起的 一个称为诱变归巢的过程，它从模板重复序列（TR）转移序列信息， 可变重复序列（VR）导致腺嘌呤到随机核苷酸的转换。远景目标 PI小组的主要任务是了解DGR诱变归巢的机制，并开发它们， 实际应用等与相关的追溯元素类似，DGR归巢被认为发生在 通过靶DNA启动的逆转录机制。有趣的是，最近的发现 PI的研究小组表明，博德特氏菌噬菌体DGR的TR的逆转录是由一个启动子启动的。 RNA中间体的下游腺嘌呤残基，并且是靶标（VR）非依赖性的。有趣的是， 发现TR RNA中间体在bRT（博德特氏菌属逆转录酶）依赖性转录因子中被切割。 以产生用于cDNA引发的3 '-OH的方式，以及在RT催化下的单个氨基酸取代 核心的破坏，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，是由于 通过bRT测定标准脱氧核糖核苷酸。此外，突变分析表明，这种特殊的， 靶非依赖性逆转录反应负责DGR诱变归巢，揭示了 DNA高变异的新机制具体的目标是基于这些新的发现。 目的1将描述TR RNA的二级结构，并定位TR RNA上的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， 揭示TR RNA中间体是否被bRT催化裂解。目标2将决定 VR的3'和5'端的cDNA整合机制。碱基配对相互作用的作用 RNA引物和VR DNA之间以及cDNA和VR DNA之间在3' cDNA整合中的相互作用将被 测试.这些研究可能导致新的cDNA整合机制的发现。目标3将 确定腺嘌呤特异性诱变的机制，这是DGRs的标志。 了解腺嘌呤特异性突变的机制可能会对RT产生新的见解 忠诚问题总之，本申请中提出的研究将阐明DGR的机制 诱变归巢，这可能在健康和科学方面具有广泛的影响。

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.