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

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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.