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

描述（由申请人提供）：最近Boshoff、Reed、巴里和Mizrahi在“细胞”中的一篇论文强烈暗示了来自M.结核分枝杆菌耐药性的形成。结核菌株。Pol E2似乎属于易错DNA聚合酶家族，并且在暴露于M.结核菌培养物对DNA损伤的条件。另一个M。tb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的成员的常规作用外，还已知参与DNA损伤修复。M的Pol I。结核病的独特之处在于它缺乏校正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都可能与M.从而在这些细菌的药物靶位点产生突变。因此，我们在E.大肠杆菌过表达载体，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敲除了M. tb和pol I（M.smeg）的敲除，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物以及巨噬细胞感染系统中产生利福平抗性基因型的相对能力。这些研究具有重要意义和及时性，但对M.因此，本次提交作为R21（探索性项目）。这两种聚合酶的表征将产生关于这些重要酶的性质的基线信息，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.