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

描述（由申请人提供）：Boshoff、Reed、Barry和Mizrahi最近在《细胞》杂志上发表的一篇论文强烈地表明，来自结核分枝杆菌的DNA聚合酶E2 （DnaE2）与结核分枝杆菌菌株的耐药性发展有关。Pol E2似乎属于一个容易出错的DNA聚合酶家族，并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存在于所有细菌物种中，除了作为后链复制机制的常规成员外，它还参与DNA损伤修复。结核分枝杆菌的Pol I在某种程度上是独特的，因为它缺乏校对3'外切酶活性，因此具有很强的致突变潜力。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点产生突变。因此，我们在大肠杆菌过表达载体上克隆了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翻译合成的能力。此外，由于dnaE2敲除结核分枝杆菌菌株和pol I敲除菌株（M.smeg）已经存在，我们想要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，以及它们在细胞培养和巨噬细胞感染系统中产生利福平耐药基因型的相对能力。这些研究具有很高的意义，而且是及时的，但是关于结核分枝杆菌的Pol E2和Pol I的初步数据很少，因此本文作为R21（探索性项目）提交。对这两种聚合酶的鉴定将产生关于这些重要酶特性的基线信息，并将验证它们作为具有高潜力的抗多药耐药结核病药物开发的有吸引力的靶点。

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.