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 他们的宿主适应不断变化的环境。高度变异是由容易出错的逆转录转座造成的 称为诱变归巢的过程，该过程从模板重复序列 (TR) 转移序列信息 可变重复序列 (VR)，导致腺嘌呤转化为随机核苷酸。长期目标 PI 小组的任务是了解 DGR 诱变归巢的机制，并将其开发用于 实际应用。与相关逆转录因子类似，DGR 归巢被提议发生 通过目标 DNA 引发的逆转录机制。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 逆转录是由 RNA 中间体的下游腺嘌呤残基，并且与靶点 (VR) 无关。有趣的是， TR RNA 中间体被发现在 bRT（博德特氏菌逆转录酶）依赖性中存在切口 生成用于 cDNA 引发的 3’-OH 的方式，并在 RT 催化下进行单氨基酸取代 核心消除了切口活性，表明bRT在裂解反应中发挥催化作用。 腺嘌呤特异性诱变发生在 (–)cDNA 合成过程中，是由于错误掺入而导致的 bRT 标准脱氧核糖核苷酸。此外，突变分析表明，这种特殊的， 不依赖靶标的逆转录反应负责 DGR 诱变归巢，揭示 DNA高度变异的新机制。具体目标基于这些新发现。 目标 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。这些研究将生成 DGR RNA 的第一个二级结构，并且 揭示 TR RNA 中间体是否被 bRT 催化裂解。目标 2 将确定 VR 3'和5'端的cDNA整合机制。碱基配对相互作用的作用 3' cDNA 整合中 RNA 引物和 VR DNA 之间以及 cDNA 和 VR DNA 之间 已测试。这些研究可能会导致新的 cDNA 整合机制的发现。目标3将 确定腺嘌呤特异性诱变机制，这是 DGR 的标志。 了解腺嘌呤特异性诱变机制可能会给 RT 带来新的见解 保真度问题。总之，本申请提出的研究将阐明 DGR 的机制 诱变归巢，这可能对健康和科学产生广泛的影响。

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.