The DNA relaxation activity and covalent complex accumulation of Mycobacterium tuberculosis topoisomerase I can be assayed in Escherichia coli: application for identification of potential FRET-dye labeling sites

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium tuberculosis </it>topoisomerase I (MtTOP1) and <it>Escherichia coli </it>topoisomerase I have highly homologous transesterification domains, but the two enzymes have distinctly different C-terminal domains. To investigate the structure-function of MtTOP1 and to target its activity for development of new TB therapy, it is desirable to have a rapid genetic assay for its catalytic activity, and potential bactericidal consequence from accumulation of its covalent complex.</p> <p>Results</p> <p>We show that plasmid-encoded recombinant MtTOP1 can complement the temperature sensitive <it>topA </it>function of <it>E. coli </it>strain AS17. Moreover, expression of MtTOP1-G116 S enzyme with the TOPRIM mutation that inhibits DNA religation results in SOS induction and loss of viability in <it>E. coli</it>. The absence of cysteine residues in the MtTOP1 enzyme makes it an attractive system for introduction of potentially informative chemical or spectroscopic probes at specific positions via cysteine mutagenesis. Such probes could be useful for development of high throughput screening (HTS) assays. We employed the AS17 complementation system to screen for sites in MtTOP1 that can tolerate cysteine substitution without loss of complementation function. These cysteine substitution mutants were confirmed to have retained the relaxation activity. One such mutant of MtTOP1 was utilized for fluorescence probe incorporation and fluorescence resonance energy transfer measurement with fluorophore-labeled oligonucleotide substrate.</p> <p>Conclusions</p> <p>The DNA relaxation and cleavage complex accumulation of <it>M. tuberculosis </it>topoisomerase I can be measured with genetic assays in <it>E. coli</it>, facilitating rapid analysis of its activities, and discovery of new TB therapy targeting this essential enzyme.</p

Population, poverty, politics and the reproductive health bill

Following an earlier paper titled 'Population and Poverty: The Real Score' (UPSE Discussion Paper 0415, December 2004), the present paper was first issued in August 2008 as a contribution to the public debate on the population issue that never seemed to die in this country. The debate heated up about that time in reaction to a revival of moves to push for legislation on reproductive health and family planning (RH/FP). Those attempts at legislation, however, failed in the 13th Congress, and again in the 14th Congress. Since late last year, the debate has been heating up further on the heels of President Noy Aquino's pronouncements seeming to favor RH/FP, though he prefers the nomenclature 'responsible parenthood'. With some updating of the data, this paper remains as relevant as ever to the ongoing public debate. It is being re-issued as a Discussion Paper for wider circulation

Inhibition of Mg2+ binding and DNA religation by bacterial topoisomerase I via introduction of an additional positive charge into the active site region

Among bacterial topoisomerase I enzymes, a conserved methionine residue is found at the active site next to the nucleophilic tyrosine. Substitution of this methionine residue with arginine in recombinant Yersinia pestis topoisomerase I (YTOP) was the only substitution at this position found to induce the SOS response in Escherichia coli. Overexpression of the M326R mutant YTOP resulted in ∼4 log loss of viability. Biochemical analysis of purified Y. pestis and E. coli mutant topoisomerase I showed that the Met to Arg substitution affected the DNA religation step of the catalytic cycle. The introduction of an additional positive charge into the active site region of the mutant E. coli topoisomerase I activity shifted the pH for optimal activity and decreased the Mg2+ binding affinity. This study demonstrated that a substitution outside the TOPRIM motif, which binds Mg2+directly, can nonetheless inhibit Mg2+ binding and DNA religation by the enzyme, increasing the accumulation of covalent cleavage complex, with bactericidal consequence. Small molecules that can inhibit Mg2+ dependent religation by bacterial topoisomerase I specifically could be developed into useful new antibacterial compounds. This approach would be similar to the inhibition of divalent ion dependent strand transfer by HIV integrase in antiviral therapy