Mediation of DNA Repair in Mycobacteria by CarD Proteins and ADP-RIbosylation

CarD 蛋白和 ADP-核糖基化介导分枝杆菌 DNA 修复

• 批准号: 7330180
• 负责人: Christina Leigh Stallings
• 金额: $ 4.68万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7330180
• 关键词: ADP ribosylation; Aerosols; Air; Animal Model; Antibiotic Resistance; Biochemistry; Biological Assay; Cessation of life; Code; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Microarray Chip; DNA Microarray format; DNA Repair; DNA strand break; Data; Development; Disease; Disease model; Drug resistance; Environment; Event; Evolution; Future; Genes; Genetic; Genome; Genus Mycobacterium; Health; Immune system; In Vitro; Infection; Investigation; Mediation; Microarray Analysis; Mission; Multi-Drug Resistance; Mutagens; Mutation; Mycobacterium Infections; Mycobacterium smegmatis; Mycobacterium tuberculosis; National Institute of Allergy and Infectious Disease; Nucleotide Excision Repair; Nutrient; Pathway interactions; Pharmaceutical Preparations; Play; Polymerase Chain Reaction; Post-Translational Protein Processing; Process; Proteins; Publishing; Rifampin; Role; Starvation; Stress; Thinking; Time; Transferase; Tuberculosis; World Health; homologous recombination; in vivo; insight; interest; killings; macrophage; mouse model; mutant; mycobacterial; novel therapeutics; pathogen; prevent; repaired; research study; response

DESCRIPTION (provided by applicant): The Mycobacterium tuberculosis health crisis is exacerbated by the alarming emergence of multi-drug resistant strains. The development of new chemotherapeutic strategies is imperative, which requires insight into the pathways involved in M. tuberculosis infection and drug resistance. Studies suggest that M. tuberculosis utilizes DNA repair to resist killing by genotoxins as well as to acquire antibiotic resistance. DNA microarrays in Mycobacterium smegmatis have implicated two proteins to be involved in regulating DNA repair: the CarD-like transcriptional regulator (CarD) and Arr ADP-ribosyl transferase (Arr). These unexplored genes stand out due to their >2 fold level of induction following DNA damage, presence in published gene sets from similar screens, and predicted functions in controlling DNA repair. Therefore, the objective of this study is to elucidate the functions of CarD and ADP-ribosylation during mycobacterial DNA damage by integrating genetics, biochemistry, and in vivo disease modeling into three specific aims. First, the roles of CarD and Arr during DNA repair will be established by analysis of M. smegmatis null mutants in genotoxic killing assays. Second, after investigating the roles of CarD and Arr during DNA damage, their respective targets will be identified. DNA microarrays will be used to gain insight into the downstream factors in CarD and Arr associated pathways. CarD transcriptional targets will be verified by in vitro DNA binding assays, while Arr post-translational modification targets will be investigated via in vitro ADP-ribosylation assays. Lastly, the functions of CarD and ADP-ribosylation during DNA damage in vitro will be confirmed in vivo by analyzing M. tuberculosis null mutants in the mouse model of infection and in cultured macrophages. These investigations will provide critical insight into the mycobacterial DNA damage response and aid in future treatments of bacterial pathogens. Mycobacteria infections have an enormous impact on world health and the objectives of this application coincide with the mission of the NIAID to better understand, treat, and ultimately prevent mycobacterial infection. Mycobacterium tuberculosis results in approximately 8 million new cases of active tuberculosis and over 2 million deaths annually. The experiments proposed will provide critical insight into the evolution of antibiotic resistance and the pathogen's ability to resist host derived DNA damage to aid in future therapies of mycobacterial disease.

描述（由申请人提供）：多药耐药菌株的惊人出现加剧了结核分枝杆菌的健康危机。开发新的化疗策略势在必行，这需要深入了解结核分枝杆菌感染和耐药性的途径。研究表明，结核分枝杆菌利用DNA修复来抵抗基因毒素的杀伤，并获得抗生素耐药性。耻垢分枝杆菌的DNA微阵列研究表明，有两种蛋白质参与DNA修复的调节：卡样转录调节因子（CarD）和Arr adp -核糖基转移酶（Arr）。这些未被探索的基因之所以突出，是因为它们在DNA损伤后的诱导水平为bb20倍，存在于来自类似筛选的已发表的基因集中，并预测了控制DNA修复的功能。因此，本研究的目的是通过将遗传学、生物化学和体内疾病建模整合为三个特定目标，阐明分枝杆菌DNA损伤过程中CarD和adp核糖基化的功能。首先，CarD和Arr在DNA修复中的作用将通过对耻毛分枝杆菌零突变体的基因毒性杀伤分析来确定。其次，在研究了CarD和Arr在DNA损伤过程中的作用后，将确定它们各自的靶点。DNA微阵列将用于深入了解CarD和Arr相关途径的下游因素。CarD转录靶点将通过体外DNA结合试验验证，而Arr翻译后修饰靶点将通过体外adp核糖基化试验进行研究。最后，通过对小鼠感染模型和培养巨噬细胞中的结核分枝杆菌零突变体的分析，在体内证实CarD和adp核糖基化在体外DNA损伤中的作用。这些研究将为分枝杆菌DNA损伤反应提供关键的见解，并有助于未来治疗细菌性病原体。分枝杆菌感染对世界卫生有着巨大的影响，这一应用程序的目标与NIAID的使命相吻合，即更好地了解、治疗并最终预防分枝杆菌感染。结核分枝杆菌每年造成大约800万活动性结核病新发病例和200多万人死亡。提出的实验将为抗生素耐药性的进化和病原体抵抗宿主来源的DNA损伤的能力提供关键的见解，以帮助分枝杆菌疾病的未来治疗。