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MECHANISM OF RECOMBINATION BY HIV REVERSE TRANSCRIPTASE

HIV逆转录酶的重组机制

基本信息

批准号：
2701621
负责人：
JEFFREY J DESTEFANO
金额：
$ 12.07万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-05-01 至 1999-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2701621
关键词：
DNA directed RNA polymerase RNA directed DNA polymerase active sites enzyme mechanism gel electrophoresis genetic recombination genetic regulatory element human immunodeficiency virus nucleic acid biosynthesis nucleic acid chemical synthesis nucleic acid sequence nucleoproteins pancreatic ribonuclease protein structure ribonuclease H transfection

项目摘要

This proposal will focus on the mechanism of human immunodeficiency virus (HIV) recombination as catalyzed by reverse transcriptase (RT). Recombination during first strand DNA synthesis (synthesis directed by the RNA genome of the retrovirus) is presumed to occur by strand transfer. This process, whereby DNA synthesized on one template (donor) is transferred to another template (acceptor) for additional elongation, occurs twice during the retroviral replication cycle. In addition to these required transfers which occur at the termini of viral templates, other transfers within internal regions of the genome can also occur. An in vitro system designed to examine internal strand transfers will be used to model recombination occurring within internal regions of the donor. Experiments will evaluate the kinetic parameters associated with this process and the influence of RT on specific steps (for example, the dissociation of the nascent DNA from the donor and its association with the acceptor) with the goal of formulating a model for recombination. Strand transfer on specific viral sequences with characteristics that may promote efficient transfer will be examined using a system that can measure the strand transfer efficiency of any region of RNA. The potential effect of base misincorporations on transfer efficiency and the frequency of misincorporation during transfer events will be evaluated. This is particularly relevant given the low fidelity of DNA synthesis by HIV-RT and the fact that misincorporation leads to DNA synthesis pausing which has been proposed to promote transfer. Nucleocapsid protein (NCp) is a nucleic acid binding protein found in the core of the retroviral virion. The properties of this protein suggest that it may influence the DNA synthetic and strand transfer catalysis properties of RT. These possibilities will be evaluated in the above systems. In addition to the studies on recombination, the RNase H cleavage specificity of HIV-RT will be examined, The experiments will use substrates that mimic structures present during replication to determine how the RT recognizes and cleaves such structures and to evaluate the spacial and topological arrangement of the polymerase and RNase H active sites of the RT. Overall, the studies will offer insight into one of the mechanisms by which retroviruses evolve and escape both drug therapy and the host immune response. An understanding of these processes is important for designing drug therapies or other strategies that target unique RT activities.

这项建议将重点放在人类免疫缺陷病毒的机制上逆转录酶(RT)催化的(HIV)重组。第一链DNA合成过程中的重组(由逆转录病毒的RNA基因组)被认为是通过链转移发生的。这一过程，即在一个模板(供体)上合成DNA是转移到另一模板(接受物)以用于额外的伸长，在逆转录病毒复制周期中发生两次。除了这些之外，在病毒模板的末端发生的所需传输，其他基因组内部区域的转移也可能发生。An In 用于检查内部链转移的体外系统将被用于模型重组发生在供体的内部区域。实验将评估与此相关的动力学参数过程和RT对特定步骤的影响(例如，供体新生DNA的分离及其与供体的关系接受者)，目标是制定一种重组模型。在特定病毒序列上进行链转移，其特征可能是促进高效转移将使用一个可以测量RNA任何区域的链转移效率。潜力碱基错位对传输效率和频率的影响将对转会活动期间的错误合并进行评估。这是特别是考虑到通过HIV-RT合成DNA的保真度较低事实上，错误的结合会导致DNA合成暂停，提出了促进转移的建议。核衣壳蛋白(NCP)是一种位于逆转录病毒粒子核心的核酸结合蛋白。这种蛋白质的性质表明它可能会影响DNA RT的合成和链转移催化性能。这些将在上述系统中评估可能性。除 HIV-RT Will重组及RNase H裂解特异性的研究实验将使用模拟结构的衬底在复制过程中存在，以确定RT如何识别和切割这样的结构，并评估空间和拓扑安排 RT的聚合酶和核糖核酸酶H活性部位。总体而言，这些研究将提供对逆转录病毒进化的机制之一的洞察并逃脱药物治疗和宿主免疫反应。一个了解这些过程对于设计药物疗法很重要。或其他针对独特RT活动的策略。