Studies of DNA Pol I and Pol E2 of M. tuberculosis

结核分枝杆菌 DNA Pol I 和 Pol E2 的研究

• 批准号: 7031984
• 负责人: MUKUND J MODAK
• 金额: $ 19.44万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-15 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031984
• 关键词: 2' 3' dideoxynucleoside; DNA damage; DNA directed DNA polymerase; DNA repair; DNA replication; Mycobacterium tuberculosis; SDS polyacrylamide gel electrophoresis; affinity chromatography; bacterial genetics; deficient growth media; drug resistance; enzyme activity; gene mutation; gene targeting; genetic strain; genome; macrophage; microorganism culture; mutagens; nucleic acid sequence; oxidative stress; protein purification; protein structure function; recombinant proteins; rifamycins; transfection /expression vector

DESCRIPTION (provided by applicant): A recent paper in 'Cell' by Boshoff, Reed, Barry and Mizrahi have strongly implicated DNA Polymerase E2 (DnaE2), from M. tb in the development of drug resistance in M. tb strains. Pol E2 appears to belong to a family of error prone DNA polymerases and is induced upon exposure of M. tb cultures to DNA damaging conditions. Another M. tb polymerase, Pol I, which is constitutively present in all bacterial species is also known to participate in DNA damage repair, besides its regular role as a member of the lagging strand replication machinery. Pol I of M. tb is somewhat unique in that it lacks a proofreading 3' exonuclease activity and hence has a strong mutagenic potential. Our hypothesis is that both Pol E2 and Pol I may be responsible for alterations in the genome sequences in M. tb thereby generating mutations at drug target sites in these bacteria. We have therefore cloned pol E2 and pol I in E. coli overexpression vectors and will purify the recombinant proteins and fully characterize these with respect to their fidelity characteristics and their ability to carry out translesion synthesis. In addition, since dnaE2 knock out strains of M. tb and knockout of pol I (M.smeg) are on hand, we would like to determine the relative survival of these strains under oxidative damage together with nutritional stress conditions and their relative ability to produce rifampicin resistance genotype in cell culture as well as in a macrophage infection system. These studies are of high significance and are timely, yet very little preliminary data on Pol E2 and Pol I from M. tb are available and hence this submission as an R21 (exploratory project). The characterization of the two polymerases will generate baseline information on the properties of these important enzymes and will validate them as attractive targets for drug development with high potential against multidrug resistant TB.

描述（由申请人提供）：Boshoff，Reed，Barry和Mizrahi在“细胞”中的最新论文强烈暗示了M. TB的DNA聚合酶E2（DNAE2），它在M. TB菌株中耐药性的发展。 Pol E2似乎属于容易发生DNA聚合酶的家族，并在M. TB培养物暴露于DNA损伤条件下引起。众所周知，在所有细菌物种中构成构成的另一个M. TB聚合酶Pol I，除了其作为滞后链复制机制的成员的常规作用外，还可以参与DNA损伤修复。 M. TB的pol I有些独特，因为它缺乏校对3'外切酶活性，因此具有很强的诱变潜力。我们的假设是，Pol E2和Pol I可能负责M. TB中基因组序列的改变，从而在这些细菌的药物靶位部位产生突变。因此，我们将大肠杆菌过表达载体的pol e2和pol i克隆过，并将净化重组蛋白，并完全表征它们的富达特性及其执行Translesion合成的能力。此外，由于DNAE2敲除了M. tb的菌株和Pol I（M.Smeg）的敲除（M.Smeg）的敲除，因此我们想确定这些菌株在氧化损伤下的相对存活以及营养应激条件以及营养应激条件以及它们在细胞培养以及巨摩托学感染系统中产生利福平抗性基因型的相对能力。这些研究具有很高的意义，并且是及时的，但是有关Pol E2和M. TB的Pol I的初步数据很少，因此可以作为R21（探索性项目）。两种聚合酶的表征将产生有关这些重要酶特性的基线信息，并将验证它们作为具有抗多药耐药性结核病潜力的药物开发的有吸引力的靶标。