DNA binding shifts the redox potential of the transcription factor SoxR

Electrochemistry measurements on DNA-modified electrodes are used to probe the effects of binding to DNA on the redox potential of SoxR, a transcription factor that contains a [2Fe-2S] cluster and is activated through oxidation. A DNA-bound potential of +200 mV versus NHE (normal hydrogen electrode) is found for SoxR isolated from Escherichia coli and Pseudomonas aeruginosa. This potential value corresponds to a dramatic shift of +490 mV versus values found in the absence of DNA. Using Redmond red as a covalently bound redox reporter affixed above the SoxR binding site, we also see, associated with SoxR binding, an attenuation in the Redmond red signal compared with that for Redmond red attached below the SoxR binding site. This observation is consistent with a SoxR-binding-induced structural distortion in the DNA base stack that inhibits DNA-mediated charge transport to the Redmond red probe. The dramatic shift in potential for DNA-bound SoxR compared with the free form is thus reconciled based on a high-energy conformational change in the SoxR–DNA complex. The substantial positive shift in potential for DNA-bound SoxR furthermore indicates that, in the reducing intracellular environment, DNA-bound SoxR is primarily in the reduced form; the activation of DNA-bound SoxR would then be limited to strong oxidants, making SoxR an effective sensor for oxidative stress. These results more generally underscore the importance of using DNA electrochemistry to determine DNA-bound potentials for redox-sensitive transcription factors because such binding can dramatically affect this key protein property

TDP1 deficiency sensitizes human cells to base damage via distinct topoisomerase I and PARP mechanisms with potential applications for cancer therapy

Base damage and topoisomerase I (Top1)-linked DNA breaks are abundant forms of endogenous DNA breakage, contributing to hereditary ataxia and underlying the cytotoxicity of a wide range of anti-cancer agents. Despite their frequency, the overlapping mechanisms that repair these forms of DNA breakage are largely unknown. Here, we report that depletion of Tyrosyl DNA phosphodiesterase 1 (TDP1) sensitizes human cells to alkylation damage and the additional depletion of apurinic/apyrimidinic endonuclease I (APE1) confers hypersensitivity above that observed for TDP1 or APE1 depletion alone. Quantification of DNA breaks and clonogenic survival assays confirm a role for TDP1 in response to base damage, independently of APE1. The hypersensitivity to alkylation damage is partly restored by depletion of Top1, illustrating that alkylating agents can trigger cytotoxic Top1-breaks. Although inhibition of PARP activity does not sensitize TDP1-deficient cells to Top1 poisons, it confers increased sensitivity to alkylation damage, highlighting partially overlapping roles for PARP and TDP1 in response to genotoxic challenge. Finally, we demonstrate that cancer cells in which TDP1 is inherently deficient are hypersensitive to alkylation damage and that TDP1 depletion sensitizes glioblastoma-resistant cancer cells to the alkylating agent temozolomide

Electrochemical Characterization of Escherichia coli Adaptive Response Protein AidB

When exposed to known DNA-damaging alkylating agents, Escherichia coli cells increase production of four DNA repair enzymes: Ada, AlkA, AlkB, and AidB. The role of three enzymes (Ada, AlkA, and AlkB) in repairing DNA lesions has been well characterized, while the function of AidB is poorly understood. AidB has a distinct cofactor that is potentially related to the elusive role of AidB in adaptive response: a redox active flavin adenine dinucleotide (FAD). In this study, we report the thermodynamic redox properties of the AidB flavin for the first time, both for free protein and in the presence of potential substrates. We find that the midpoint reduction potential of the AidB flavin is within a biologically relevant window for redox chemistry at −181 mV, that AidB significantly stabilizes the flavin semiquinone, and that small molecule binding perturbs the observed reduction potential. Our electrochemical results combined with structural analysis allow for fresh comparisons between AidB and the homologous acyl-coenzyme A dehydrogenase (ACAD) family of enzymes. AidB exhibits several discrepancies from ACADs that suggest a novel catalytic mechanism distinct from that of the ACAD family enzymes.National Institutes of Health (U.S.) (Grant P30-ES002109)National Institutes of Health (U.S.) (Grant R01-GM69857)National Science Foundation (U.S.) (Grant MCB-0543833

The redox state of the [2Fe-2S] clusters in SoxR protein regulates its activity as a transcription factor

SoxR protein is a redox-responsive transcription factor that governs a regulon of oxidative stress and antibiotic resistance genes in Escherichia coli. Purified SoxR contains oxidized [2Fe-2S] clusters and stimulates in vitro transcription of its target gene soxS up to 100-fold. SoxR transcriptional activity, but not DNA binding, is completely dependent on the [2Fe-2S] clusters; apo-SoxR prepared in vitro binds the soxS promoter with unchanged affinity but does not have transcription activity. Thus, modulation of the SoxR [2Fe-2S] clusters was proposed to control the protein\u27s function in transcription. Here, we provide evidence that SoxR with reduced [2Fe-2S] clusters is inactive. Redox titration of purified SoxR revealed a midpoint potential of -285 ± 10 mV (pH 7.6). In vitro transcription assays showed that SoxR was inactivated when the [2Fe-2S] cluster was reduced (-380 mV), and full activity was restored upon reoxidation (+100 mV). The results suggest that one-electron oxidation and reduction of the [2Fe-2S] cluster regulate SoxR transcriptional activity

Molecular characterization of an adaptive response to alkylating agents in the opportunistic pathogen Aspergillus fumigatus.

An adaptive response to alkylating agents based upon the conformational change of a methylphosphotriester (MPT) DNA repair protein to a transcriptional activator has been demonstrated in a number of bacterial species, but this mechanism appears largely absent from eukaryotes. Here, we demonstrate that the human pathogen Aspergillus fumigatus elicits an adaptive response to sub-lethal doses of the mono-functional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We have identified genes that encode MPT and O(6)-alkylguanine DNA alkyltransferase (AGT) DNA repair proteins; deletions of either of these genes abolish the adaptive response and sensitize the organism to MNNG. In vitro DNA repair assays confirm the ability of MPT and AGT to repair methylphosphotriester and O(6)-methylguanine lesions respectively. In eukaryotes, the MPT protein is confined to a select group of fungal species, some of which are major mammalian and plant pathogens. The evolutionary origin of the adaptive response is bacterial and rooted within the Firmicutes phylum. Inter-kingdom horizontal gene transfer between Firmicutes and Ascomycete ancestors introduced the adaptive response into the Fungal kingdom. Our data constitute the first detailed characterization of the molecular mechanism of the adaptive response in a lower eukaryote and has applications for development of novel fungal therapeutics targeting this DNA repair system

John B. Little Center Annual Symposium

The Annual Symposium of the John B. Little Center for Radiation Sciences and Environmental Health at the Harvard School of Public Health seeks to educate radiobiologists and biomedical scientists in related areas on the leading research related to the effects of ionizing radiation and related environmental agents in biological systems. This effort seeks to further the training of individuals in this field, and to foment productive interactions and collaborations among scientists at Harvard and with other institutions. The Symposium attracts world-class scientists as speakers, and a broad cross-section of attendees from academic, government, and industrial research centers, as well as editorial staff from leading scientific publications. In order to maintain this quality, funding to support the travel and local expenses of invited speakers is sought, along with funds to allow use of appropriate conference facilities