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 最近在《Cell》上发表的一篇论文强烈暗示结核分枝杆菌 DNA 聚合酶 E2 (DnaE2) 与结核分枝杆菌菌株耐药性的发展有关。 Pol E2 似乎属于容易出错的 DNA 聚合酶家族，并且在结核分枝杆菌培养物暴露于 DNA 损伤条件时被诱导。另一种结核分枝杆菌聚合酶 Pol I 组成型存在于所有细菌物种中，除了作为滞后链复制机制成员的常规作用外，还已知它参与 DNA 损伤修复。 M. tb 的 Pol I 有点独特，因为它缺乏校对 3' 核酸外切酶活性，因此具有很强的诱变潜力。我们的假设是，Pol E2 和 Pol I 都可能导致结核分枝杆菌基因组序列的改变，从而在这些细菌的药物靶位点产生突变。因此，我们在大肠杆菌过表达载体中克隆了 pol E2 和 pol I，并将纯化重组蛋白并充分表征它们的保真度特征和进行跨损伤合成的能力。此外，由于 dnaE2 敲除 M. tb 菌株和敲除 pol I (M.smeg) 菌株已经存在，我们希望确定这些菌株在氧化损伤和营养应激条件下的相对存活率及其相对能力在细胞培养物以及巨噬细胞感染系统中产生利福平抗性基因型。这些研究意义重大且及时，但关于 M. tb 的 Pol E2 和 Pol I 的初步数据很少，因此本次提交为 R21（探索性项目）。这两种聚合酶的表征将生成有关这些重要酶特性的基线信息，并将验证它们作为药物开发的有吸引力的目标，具有对抗多重耐药结核病的巨大潜力。