DNA repair and recombination within the var gene family of P. falciparum

描述(申请人提供)：疟疾是由疟原虫属原生动物寄生虫引起的。恶性疟原虫是毒性最强的物种，是大多数发病率和死亡率的罪魁祸首，包括每年主要发生在幼儿中的约80万人死亡。寄生虫能够在宿主免疫系统的攻击下幸存下来的关键是它修复DNA损伤的能力。DNA双链断裂(DSB)如果不修复是致命的，疟疾寄生虫似乎缺少大多数真核生物修复这种断裂的两条主要途径之一。此外，剩余的途径需要与断裂位置同源的第二个DNA拷贝作为修复的模板。考虑到疟疾寄生虫在其生命周期的大部分时间都是单倍体，因此大多数基因组都是作为单一副本保持的，它们如何修复DSB仍然是一个谜。这项提议的长期目标是了解寄生虫如何维持其基因组的完整性，并确定DNA修复途径如何有助于编码其主要表面抗原的基因的多样化。为了达到这些目标，一种可调节的、位点特异的内切酶系统已被改造用于培养的寄生虫。该系统能够在基因组的目标位置诱导单个DSB，然后快速有效地分离修复产物。在项目的第一个人工智能中，这个系统将被用来描述寄生虫修复DSB的基本机制，包括这个过程有多容易出错，是否需要模板，以及错配修复途径在DSB修复中的作用。在第二个目标中，该系统将用于研究var基因家族的多样性。Var基因编码PfEMP1，这是疟疾的主要毒力因子。这个庞大的、多拷贝的基因家族经历了快速和持续的多样化，使寄生虫能够通过抗原变异避开免疫系统。多样性背后的机制尚不清楚，但这一过程似乎涉及基因片段的频繁“洗牌”，这是DSB修复的产物基因转换事件的一个标志。位点特异性内切酶系统将被用来确定DSB修复如何有助于var基因的多样化。 与公共卫生相关：疟疾寄生虫利用其基本的DNA修复途径在人类免疫反应造成的DNA损伤中存活下来，并在受感染的红细胞表面表达的编码蛋白质的基因中产生多样性。对寄生虫如何能够避免免疫或确定寄生虫修复DNA损伤能力的潜在弱点的调查将有助于开发新的疾病干预策略。

DESCRIPTION (provided by applicant): Malaria is caused by protozoan parasites of the genus Plasmodium. P. falciparum is the most virulent species and is responsible for the majority of both morbidity and mortality, including approximately 800,000 deaths per year that occur mainly among young children. Key to the parasite's ability to survive attack by the host's immune system is its ability to repair DNA damage. DNA double strand breaks (DSBs) are lethal if not repaired, and malaria parasites appear to be missing one of the two primary pathways used by most eukaryotes to repair such breaks. In addition, the remaining pathway requires a second DNA copy homologous to the site of the break to serve as a template for repair. Considering that malaria parasites are haploid for most of their lifecycle and therefore the majority of the genome is maintained as a single copy, how they repair DSBs remains a mystery. The long-term objectives of this proposal are to understand how parasites maintain the integrity of their genomes, and to determine how DNA repair pathways contribute to diversification of the genes encoding their primary surface antigens. To address these objectives, a regulatable, site-specific endonuclease system has been adapted for use in cultured parasites. This system enables the induction of a single DSB within a targeted site of the genome followed by the rapid and efficient isolation of the products of repair. In the first ai of the project, this system will be used to characterize the basic mechanisms employed by parasites to repair DSBs, including how error prone the process is, whether a template is required, and the role of the mismatch repair pathway in DSB repair. In the second aim, the system will be applied to investigating diversification of the var gene family. var genes encode PfEMP1, the primary malaria virulence factor. This large, multi-copy gene family undergoes rapid and continuous diversification that enables the parasite to avoid the immune system through antigenic variation. The mechanisms underlying diversification are unknown, but the process appears to involve frequent "shuffling" of segments, a hallmark of gene conversion events that are a product of DSB repair. The site-specific endonuclease system will be used to determine how DSB repair contributes to var gene diversification. PUBLIC HEALTH RELEVANCE: Malaria parasites utilize their basic DNA repair pathways both to survive DNA damage resulting from the human immune response and to generate diversity within the genes encoding proteins expressed on the surface of infected red blood cells. Investigations into how the parasite is able to avoid immunity or identify potential weaknesses in the parasite's ability to repair DNA damage will contribute to the development of novel disease intervention strategies.