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

Human DNA polymerase β polymorphism, Arg137Gln, impairs its polymerase activity and interaction with PCNA and the cellular base excision repair capacity

DNA polymerase β (Pol β) is a key enzyme in DNA base excision repair, and an important factor for maintaining genome integrity and stability. More than 30% of human tumors characterized to date express DNA Pol β variants, many of which result from a single nucleotide residue substitution. However, in most cases, their precise functional deficiency and relationship to cancer susceptibility are still unknown. In the current work, we show that a polymorphism encoding an arginine to glutamine substitution, R137Q, has lower polymerase activity. The substitution also affects the interaction between Pol β and proliferating cell nuclear antigen (PCNA). These defects impair the DNA repair capacity of Pol β in reconstitution assays, as well as in cellular extracts. Expression of wild-type Pol β in pol β−/− mouse embryonic fibroblast (MEF) cells restored cellular resistance to DNA damaging reagents such as methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (MNU), while expression of R137Q in pol β−/− MEF cells failed to do so. These data indicate that polymorphisms in base excision repair genes may contribute to the onset and development of cancers

Flavin-Induced Oligomerization in Escherichia coli Adaptive Response Protein AidB

The process known as “adaptive response” allows Escherichia coli to respond to small doses of DNA-methylating agents by upregulating the expression of four proteins. While the role of three of these proteins in mitigating DNA damage is well understood, the function of AidB is less clear. Although AidB is a flavoprotein, no catalytic role has been established for the bound cofactor. Here we investigate the possibility that flavin plays a structural role in the assembly of the AidB tetramer. We report the generation and biophysical characterization of deflavinated AidB and of an AidB mutant that has greatly reduced affinity for flavin adenine dinucleotide (FAD). Using fluorescence quenching and analytical ultracentrifugation, we find that apo AidB has a high affinity for FAD, as indicated by an apparent dissociation constant of 402.1 ± 35.1 nM, and that binding of substoichiometric amounts of FAD triggers a transition in the AidB oligomeric state. In particular, deflavinated AidB is dimeric, whereas the addition of FAD yields a tetramer. We further investigate the dimerization and tetramerization interfaces of AidB by determining a 2.8 Å resolution crystal structure in space group P32 that contains three intact tetramers in the asymmetric unit. Taken together, our findings provide strong evidence that FAD plays a structural role in the formation of tetrameric AidB.National Institutes of Health (U.S.) (grant R01-GM0272663)National Institutes of Health (U.S.) (grant P30-ES002109)National Science Foundation (U.S.) (grant MCB-0543833

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins

Characterization of magnesium requirement of human 5'-tyrosyl DNA phosphodiesterase mediated reaction

<p>Abstract</p> <p>Background</p> <p>Topo-poisons can produce an enzyme-DNA complex linked by a 3'- or 5'-phosphotyrosyl covalent bond. 3'-phosphotyrosyl bonds can be repaired by tyrosyl DNA phosphodiesterase-1 (TDP1), an enzyme known for years, but a complementary human enzyme 5'-tyrosyl DNA phosphodiesterase (hTDP2) that cleaves 5'-phosphotyrosyl bonds has been reported only recently. Although hTDP2 possesses both 3'- and 5'- tyrosyl DNA phosphodiesterase activity, the role of Mg²⁺in its activity was not studied in sufficient details.</p> <p>Results</p> <p>In this study we showed that purified hTDP2 does not exhibit any 5'-phosphotyrosyl phosphodiesterase activity in the absence of Mg²⁺/Mn²⁺, and that neither Zn²⁺or nor Ca²⁺can activate hTDP2. Mg²⁺also controls 3'-phosphotyrosyl activity of TDP2. In MCF-7 cell extracts and de-yolked zebrafish embryo extracts, Mg²⁺controlled 5'-phosphotyrosyl activity. This study also showed that there is an optimal Mg²⁺concentration above which it is inhibitory for hTDP2 activity.</p> <p>Conclusion</p> <p>These results altogether reveal the optimal Mg²⁺requirement in hTDP2 mediated reaction.</p