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

描述（由申请人提供）：疟疾是由疟原虫属的原生动物寄生虫引起的。 恶性疟原虫是毒性最强的物种，是造成大部分发病和死亡的原因，其中每年约 800,000 例死亡，主要发生在幼儿中。寄生虫能够抵抗宿主免疫系统攻击的关键是其修复 DNA 损伤的能力。 DNA 双链断裂 (DSB) 如果不修复，就会致命，而疟疾寄生虫似乎缺失了大多数真核生物修复此类断裂的两条主要途径之一。此外，剩余的途径需要与断裂位点同源的第二个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里都是单倍体，因此大部分基因组都保持为单个拷贝，它们如何修复 DSB 仍然是一个谜。该提案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 修复途径如何促进编码其主要表面抗原的基因的多样化。为了实现这些目标，可调节的、位点特异性核酸内切酶系统已适用于培养的寄生虫。该系统能够在基因组的目标位点内诱导单个 DSB，然后快速有效地分离修复产物。在该项目的第一个ai中，该系统将用于表征寄生虫修复DSB的基本机制，包括该过程的出错率、是否需要模板以及错配修复途径在DSB修复中的作用。第二个目标是，该系统将应用于研究 var 基因家族的多样化。 var 基因编码 PfEMP1，即主要的疟疾毒力因子。这个庞大的多拷贝基因家族经历快速且持续的多样化，使寄生虫能够通过抗原变异来躲避免疫系统。多样化背后的机制尚不清楚，但该过程似乎涉及片段的频繁“洗牌”，这是 DSB 修复产物基因转换事件的标志。位点特异性核酸内切酶系统将用于确定 DSB 修复如何促进 var 基因多样化。

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.