Integration and Excision by Serine Intergrases

描述(申请人提供)：基因重组是一个基本的生物学过程，发生在几乎所有的生物体中。位点特异性重组是一类由作用于特定DNA位点的特殊酶介导的重组事件，可催化广泛的生物学结果，包括病毒整合、细菌抗原切换和免疫多样性的产生。这项建议的目的是了解位点特异性重组的机制和调控。我们研究的模型系统是分枝杆菌噬菌体Bxb1的整合和切除，这是一种感染分枝杆菌宿主的病毒。这些寄主细菌具有相当重要的医学价值，包括结核分枝杆菌和麻风分枝杆菌，它们分别是人类结核病和麻风的病原体。它们的操作所需的遗传系统仍然很不完善，分支杆菌噬菌体的特性在改进疫苗、新药和更快的诊断工具的开发方面具有巨大的潜力。分枝杆菌噬菌体Bxb1的整合和切割系统是不寻常的，因为这些重组事件是由丝氨酸重组酶家族的整合酶蛋白催化的。这些重组酶是较新发现的，初步分析表明，它们的作用机制明显不同于已知的酪氨酸重组酶整合酶。更具体地说，Bxb1整合酶作用于重组两个DNA位点-attB和attP-这两个DNA位点很小(&lt；50bp)，在序列和大小上不同，并且不需要其他DNA或蛋白质成分。然而，这种酶很聪明，它只会重组这些位点，以产生attL和Attr产物；它不会重组任何其他位点组合。尽管如此，噬菌体Bxb1编码了第二个蛋白质gp47，它指示gpInt作用于不同的位点对-attL和AttR-来介导原噬菌体切除。这代表了蛋白质功能中两种可供选择的底物之间的有趣的分子开关，并与理解自然界中的其他分子开关有关。简单性、方向性和高度特异性的靶向性使这些重组反应非常适合于在异源遗传系统中工作，包括在其他细菌病原体，疟疾，蠕虫，果蝇，哺乳动物细胞培养和小鼠中。因此，理解和操纵Bxb1系统将对几乎所有生物模式生物的遗传学产生广泛影响。 公共卫生相关性：特定部位重组机制的阐明将促进结核病新疫苗的开发，这些疫苗是操纵用于研究人类发育、癌症和疟疾等传染病的模式生物的强大工具。重组是一个核心的生物学过程，了解它的机制和调控将对我们理解生物学和医学产生广泛的影响。

DESCRIPTION (provided by applicant): Genetic recombination is a fundamental biological process that occurs in virtually all organisms. Site-specific recombination is a class of recombination events that are mediated by specialized enzymes acting at specific DNA sites, and catalyze a broad array of biological outcomes includes viral integration, bacterial antigen switching, and generation of immunological diversity. The goal of this proposal is to understand the mechanism and regulation of site-specific recombination. Our model system for investigation is the integration and excision of mycobacteriophage Bxb1, a virus that infects mycobacterial hosts. These host bacteria are of considerable medical importance, and include Mycobacterium tuberculosis and Mycobacterium leprae, the causative agents of human tuberculosis and leprosy respectively. The genetic systems required for their manipulation remains rudimentary and mycobacteriophage characterization has great potential for contributing to the development of improved vaccines, new drugs, and speedier diagnostic tools. The integration and excision system of mycobacteriophage Bxb1 is unusual in that these recombination events are catalyzed by an integrase protein that is a member of the family of serine-recombinases. These recombinases were identified relatively recently, and preliminary analysis shows that their mechanism is distinctly different to the well-characterized tyrosine-recombinase integrases. More specifically, the Bxb1 integrase acts to recombine two DNA sites - attB and attP - that are small (<50bp), different in sequence and size, and requires no other DNA or protein components. The enzyme is clever though, and will recombine only these sites, to generate attL and attR products; it will not recombine any other site combination. Nonetheless, phage Bxb1 encodes a second protein, gp47, that instructs gpInt to act on a different site pair - attL and attR - to mediate prophage excision. This represents an interesting molecular switch in protein function between two alternative choices of substrate, and is relevant to understanding other molecular switches in nature. The simplicity, directionality, and highly specific targeting, makes these recombination reactions highly suitable for adaptation to work in heterologous genetic systems including in other bacterial pathogens, malaria, in worms, fruit flies, mammalian cell culture, and in mice. Understanding and manipulating the Bxb1 system will therefore have a broad impact on the genetics of virtually all biological model organisms. PUBLIC HEALTH RELEVANCE: Elucidation of site-specific recombination mechanisms will facilitate the development of new vaccines for tuberculosis, powerful tools for manipulating the model organisms used to study human development, cancer, and infectious diseases such as malaria. Recombination is a core biological process and understanding its mechanisms and regulation will have a broad impact on our understanding of biology and medicine.