In this study, we investigated the role of the nucleotide excision repair (NER) pathway in mycobacterial DNA repair. Mycobacterium smegmatis lacking the NER excinuclease component uvrB or the helicase uvrD1 gene and a double knockout lacking both genes were constructed, and their sensitivities to a series of DNA-damaging agents were analyzed. As anticipated, the mycobacterial NER system was shown to be involved in the processing of bulky DNA adducts and interstrand cross-links. In addition, it could be shown to exert a protective effect against oxidizing and nitrosating agents. Interestingly, inactivation of uvrB and uvrD1 significantly increased marker integration frequencies in gene conversion assays. This implies that in mycobacteria (which lack the postreplicative mismatch repair system) NER, and particularly the UvrD1 helicase, is involved in the processing of a subset of recombination-associated mismatches.The success of Mycobacterium tuberculosis as a human pathogen lies to some extent in its ability to survive and replicate in macrophages (23). It is currently unknown how it manages to overcome the assault on its DNA by macrophagegenerated reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), which represent an otherwise very effective defense against intracellular pathogens (5,8,35). One possibility is that M. tuberculosis effectively detoxifies ROI and RNI. Alternatively, it may possess highly effective DNA repair machineries. In silico analyses of mycobacterial genomes including M. tuberculosis (6, 34), Mycobacterium leprae (7), Mycobacterium bovis (15), Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium smegmatis (The Institute for Genome Research [http://www.tigr.org]) revealed the presence of genes encoding enzymes involved in DNA damage reversal, nucleotide excision repair (NER), base excision repair (BER), recombinational repair, nonhomologous end joining, and SOS repair. Surprisingly, mycobacteria are devoid of the mutLS-based postreplicative mismatch repair (MMR) system (34, 51), which is otherwise highly conserved throughout evolution and contributes to mutation avoidance by correcting replication errors resulting from nucleotide misincorporation and polymerase slippage (27,46). In addition, MMR inhibits recombination between nonidentical (homeologous) sequences and thus helps to control the fidelity of recombination (32, 41, 56).The finding that mycobacteria exhibit a general mutation rate comparable to that of MMR-proficient species suggests that they possess alternative or compensating strategies for mismatch recognition and MMR (51). It is conceivable, for example, that their replication and recombination fidelity is higher than in other prokaryotes. Alternatively, it is possible that the fidelity of these processes is controlled by one of the other DNA metabolic pathways, such as NER.Prokaryotic NER has been extensively studied in Escherichia coli. It is mediated by the UvrABC excinuclease enzyme complex and the helicase UvrD (43), a system cap...

Induction of erythropoietin (Epo) expression under hypoxic conditions is mediated by the heterodimeric hypoxia-inducible factor (HIF)-1. Following binding to the 3′ hypoxia-response element (HRE) of the Epo gene, HIF-1 markedly enhances Epo transcription. To facilitate the search for HIF-1 (ant)agonists, a hypoxia-reporter cell line (termed HRCHO5) was constructed containing a stably integrated luciferase gene under the control of triplicated heterologous HREs. Among various agents tested, we identified a class of substances called epolones, which induced HRE-dependent reporter gene activity in HRCHO5 cells. Epolones are fungal products known to induce Epo expression in hepatoma cells. We found that epolones (optimal concentration 4-8 μmol/L) potently induce HIF-1α protein accumulation and nuclear translocation as well as HIF-1 DNA binding and reporter gene transactivation. Interestingly, the activity of a compound related to the fungal epolones, ciclopirox olamine (CPX), was blocked after addition of ferrous iron. This suggests that CPX might interfere with the putative heme oxygen sensor, as has been proposed for the iron chelator deferoxamine mesylate (DFX). However, about 10-fold higher concentrations of DFX (50-100 μmol/L) than CPX were required to maximally induce reporter gene activity in HRCHO5 cells. Moreover, structural, functional, and spectrophotometric data imply a chelator:iron stoichiometry of 1:1 for DFX but 3:1 for CPX. Because the iron concentration in the cell culture medium was determined to be 16 μmol/L, DFX but not CPX function can be explained by complete chelation of medium iron. These results suggest that the lipophilic epolones might induce HIF-1α by intracellular iron chelation.

Induction of erythropoietin (Epo) expression under hypoxic conditions is mediated by the heterodimeric hypoxia-inducible factor (HIF)-1. Following binding to the 3′ hypoxia-response element (HRE) of the Epo gene, HIF-1 markedly enhances Epo transcription. To facilitate the search for HIF-1 (ant)agonists, a hypoxia-reporter cell line (termed HRCHO5) was constructed containing a stably integrated luciferase gene under the control of triplicated heterologous HREs. Among various agents tested, we identified a class of substances called epolones, which induced HRE-dependent reporter gene activity in HRCHO5 cells. Epolones are fungal products known to induce Epo expression in hepatoma cells. We found that epolones (optimal concentration 4-8 μmol/L) potently induce HIF-1α protein accumulation and nuclear translocation as well as HIF-1 DNA binding and reporter gene transactivation. Interestingly, the activity of a compound related to the fungal epolones, ciclopirox olamine (CPX), was blocked after addition of ferrous iron. This suggests that CPX might interfere with the putative heme oxygen sensor, as has been proposed for the iron chelator deferoxamine mesylate (DFX). However, about 10-fold higher concentrations of DFX (50-100 μmol/L) than CPX were required to maximally induce reporter gene activity in HRCHO5 cells. Moreover, structural, functional, and spectrophotometric data imply a chelator:iron stoichiometry of 1:1 for DFX but 3:1 for CPX. Because the iron concentration in the cell culture medium was determined to be 16 μmol/L, DFX but not CPX function can be explained by complete chelation of medium iron. These results suggest that the lipophilic epolones might induce HIF-1α by intracellular iron chelation.