Novel components of Homologous Recombination Repair in the parasite Toxoplasma gondii

弓形虫寄生虫中同源重组修复的新成分

基本信息

批准号：
9906165
负责人：
Sergio Oscar Angel
金额：
$ 12.79万
依托单位：
INSTITUTE/RESEARCH/BIOTECHNOLOGY FDN
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-07 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9906165
关键词：
Acute Acute Disease Aftercare Antibodies Area BRCA1 gene BRCA2 gene BRCT Domain Biological Biological Assay Biology Bioterrorism CRISPR/Cas technology Camptothecin Cancerous Category B pathogen Cell Survival Cells Centers for Disease Control and Prevention (U.S.)Co-Immunoprecipitations Collaborations Complex Congenital Abnormality Cyst Cytolysis DNA DNA Damage DNA Double Strand Break DNA Markers DNA Repair DNA biosynthesis DNA lesion DNA replication fork Defect Development Disease Double Strand Break Repair Drug Targeting Endonuclease I Eukaryota Future G2 Phase Gamma Rays Gene Silencing Generations Genes Genetic Transcription Goals Growth Human Immunocompromised Host Impairment Individual Infection Invaded Investigation Knowledge Laboratories Life Link Lytic Phase Mammals Methodology Methods Microscopy Modeling Names Neurologic Nonhomologous DNA End Joining Parasites Pathway interactions Patients Performance Pharmaceutical Preparations Phosphotransferases Plasmids Play Process Proteins RAD9A gene Research Role S Phase Schizophrenia Stress Structure Therapeutic Tissues Topoisomerase-I Inhibitor Toxoplasma Toxoplasma gondii Toxoplasmosis Transfection United States National Institutes of Health Yeasts acute infection asexual ataxia telangiectasia mutated protein base bioinformatics tool cancer therapy chronic infection design endodeoxyribonuclease SceI genotoxicity high throughput screening homologous recombination in silico in vivo inhibitor/antagonist innovative technologies insight nervous system disorder new therapeutic target novel novel therapeutics pathogen priority pathogen protein crosslink recombinational repair recruit repaired response segregation sensor tool tumor

项目摘要

The protozoan parasite Toxoplasma gondii is an important human and veterinary pathogen. In humans, acute disease is caused by rapidly growing `tachyzoites' stage. In this context, we hypothesize that during this intense tachyzoite propagation, replication-associated DNA stress could appear to be generating fork collapse and DNA double strand breaks which should be repaired by homologous recombination repair (HRR). The hypothesis is supported by the presence of basal γH2A.X, a marker of DSB, in the tachyzoite as well as the inhibition of its replication by the KU55933, an inhibitor of ATM, a key kinase in the HRR pathway. In addition, our analysis indicates that key molecules seem to be not present in HRR pathway in T. gondii. Differences in the HRR cascade imply an opportunity for the identification of novel and specific HRR components in T. gondii. If available, such factors could represent attractive candidates for the development of new drugs against Toxoplasmosis. Our goal is to identify novel components of HRR and analyze their importance in HRR pathway. In specific aim 1 we will develop a methodology named iPOND to pull down components of HRR in T. gondii and their identification and analysis by bioinformatics tools. In addition, we will analyze their biological significance in tachyzoite replication and DSB sensitivity, together with some HRR components yet identified by in silico analysis (Mre11, RAD51, ATM, BRCA2, BRCT domain containing proteins). In specific aim 2 we will design a model of HR in vivo by generating tachyzoites conditionally expressing the endonuclease I-SceI to generate DSB specifically in a plasmid incorporated by transfection. This plasmid contains two versions of GFP inactive genes in which the functional gene is expressed only after HR.

原生动物寄生虫弓形虫弓形虫是重要的人类和兽医病原体。在人类中，急性疾病是由快速生长的“速二族人”阶段引起的。在这种情况下，我们假设在这种强烈的tachyzoite繁殖过程中，与复制相关的DNA应激似乎可能正在产生叉子塌陷，而DNA双链断裂应通过同源重组修复（HRR）来修复。在tachyzoite中的基础γH2A.x（DSB的标记基底）以及抑制其复制的KU555933（ATM的抑制剂）（HRR途径中的关键激酶）的抑制作用。此外，我们的分析表明，在T. gondii的HRR途径中，关键分子似乎不存在。 HRR级联反应的差异意味着可以识别T. gondii中新颖和特定的HRR成分的机会。如果有的话，这些因素可能代表有吸引力的候选者，用于开发针对毒质剂的新药。我们的目标是识别HRR的新成分，并分析其在HRR途径中的重要性。在特定目标1中，我们将开发一种名为IPOND的方法，以降低T. gondii中HRR的组成部分及其通过生物信息学工具的识别和分析。此外，我们将分析它们在速氮岩复制和DSB敏感性中的生物学意义，以及一些HRR成分，但通过计算机分析（MRE11，RAD51，ATM，BRCA2，含有蛋白质的BRCT结构域）鉴定出来。在特定目标2中，我们将通过有条件表达核酸内切酶I-SCEI的速度量来设计一个人力资源模型，以在翻译掺入的质粒中生成DSB。该质粒包含两个版本的GFP非活性基因，其中仅在HR后表达功能基因。