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》上发表的一篇论文强烈指出，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，当结核分枝杆菌培养物暴露在DNA破坏条件下时被诱导。另一种结核分枝杆菌聚合酶POLI存在于所有细菌物种中，除了作为滞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PolI在某种程度上是独一无二的，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，我们已在大肠杆菌超表达载体中克隆了polE2和polI，并将对重组蛋白进行纯化，并对其保真度特征和进行跨损伤合成的能力进行全面鉴定。此外，由于dna E2敲除结核分枝杆菌和敲除polI(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，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的相对能力。这些研究具有很高的意义，而且是及时的，但关于结核分枝杆菌的POL E2和POL I的初步数据非常少，因此本报告作为R21(探索性项目)提交。这两种聚合酶的特性将产生关于这些重要酶性质的基线信息，并将证实它们是具有高潜力治疗多药耐药结核病的有吸引力的药物开发目标。

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.