<p> Hydrogen sulfide (H₂S) is more commonly known for its toxic properties; however, recently, there has been evidence that this small, gaseous molecule could serve as an endogenous cell-signaling agent. Surprisingly, a number of studies have also provided evidence that H₂S is a DNA-damaging mutagen. Using a plasmid-based DNA strand cleavage assay, we examined the chemical mechanisms of DNA damage by H₂S. We found single-strand DNA cleavage was caused by micromolar concentrations of H₂S. The mechanistic process was studied and was shown to involve the autoxidation of H₂S to generate superoxide, hydrogen peroxide, and ultimately hydroxyl radical, a well-known DNA-damaging agent, via a trace metal-mediated Fenton-type reaction. In the presence of physiological thiol concentrations, DNA strand cleavage by H₂S still occurred. The oxidation byproducts of H₂S, such as thiosulfate, sulfite, and sulfate, do not contribute to DNA strand cleavage. However, the initially generated oxidation products, like persulfide (S₂^2-), most likely go through rapid autoxidation reactions, which contribute to superoxide generation. This autoxidation process is of potential relevance to both the genotoxic and cell signaling properties of H₂S.</p>

<p> Metallothioneins (MT) are low molecular weight, cysteine-rich, metal-binding proteins that are thought to play key roles in detoxification and Zn homeostasis. Four metallothionein isoform families, termed MT 1-4, have been identified in mammalian species. The majority of studies on MT have focused on the biochemical properties of the widely expressed MT-1 and MT-2 and, in comparison, few studies have investigated the metal binding characteristics of the neuronal-specific MT-3. While the function of MT-3 in neurons is not fully understood, a better understanding of the biochemical properties of MT-3 and its relationship with the other MT isoforms may aid in determining the role of this isoform in Zn homeostasis in the brain. In the current study, different MT isoforms, each labeled with a unique stable isotope of Zn, have been used to study the interaction of Zn between the MT isoforms and to study their ability to donate metal in transfer reactions.</p>

<p> DNA damage is a ubiquitous challenge to replication in cells, as damage causes replicative polymerase stalling. However, once DNA has been unwound at the replication fork, replication must proceed in the presence of damage to prevent more deleterious and almost assuredly mutagenic consequences. Alleviation of replicative polymerase stalling is accomplished by specialized DNA polymerases that can synthesize across from DNA lesions using the damage as a template, a process termed translesion synthesis (TLS). DNA polymerase &eta; pol &eta; is the best understood of these polymerases, and lack of pol &eta; synthesis activity in the human cancer prone syndrome Xeroderma pigmentosum variant (XPV) leads to cancer susceptibility upon sunlight exposure. XPV cells display higher mutation rates when exposed to UV light. This prevention of mutagenesis occurs despite pol &eta; having fidelity that is thousands of fold lower than replicative polymerases when copying both damaged and undamaged DNA. Pol &eta; has been implicated in replication past the UV induced <i> cis-syn</i> cyclobutane pyrimidine dimers (CPD) and the oxidative lesion 7,8-dihydro-8-oxo-guanine (8-oxoG) in cells. We sought to better understand the molecular basis of efficient but moderate to low fidelity bypass by pol &eta;. We have examined polymerase properties as well as replication fidelity opposite these 2 lesions and with undamaged DNA. </p><p> To this end, we have created and purified a set of single amino acid substitution mutants in and surrounding the active site of the protein, utilizing the truncated catalytic core of the protein as a model. We assessed these mutants for overall synthesis activity as well as bypass fidelity opposite both T-T CPD and 8-oxoG. Our results show that several residues are critical for polymerase function, and altering these amino acids have multiple effects on polymerase properties. The R55A mutant abolishes polymerase activity while the Q38A, Y52E, and R61A mutants display altered fidelities. Y52E increased fidelity at both lesions and undamaged DNA, while R61A increased fidelity when copying T-T CPD. Also notable, Q38A increased fidelity opposite 8-oxoG, while it decreased fidelity opposite a T-T CPD. </p><p> One proposed means of increasing pol &eta; fidelity is interaction with replication accessory proteins that assist in replication at the replication fork. We purified the full-length form of pol &eta;, containing known protein:protein interaction domains in the C-terminus, and examined the effect of adding RPA to the bypass reaction. We saw no change in fidelity when examining fidelity opposite T-T CPD or 8-oxoG. We sought to confirm our results by also expressing two previously identified mutants with specific fidelity signatures, Q38A and Y52E. These full-length mutants recapitulated the fidelity effects seen in the truncated mutants when copying damaged DNA, and these fidelity signatures were unchanged with the addition of RPA. </p><p> Taken together, these results indicate that the major determinant of pol &eta; fidelity is the active site structure of the protein. The active site sequence is robust and certain amino acids play a critical role in the molecular mechanism of synthesis by the enzyme. Further clues as to the effects of altered polymerase function could be addressed by experiments expressing these mutant proteins in cells lacking pol &eta;. Additional investigation is necessary to recapitulate a more complete set of proteins with known functions in TLS, as interaction with other proteins could possibly alter fidelity. This work emphasizes that TLS is a damage tolerance process that could potentially cause mutations if perturbed. In order to avoid the certainly mutagenic consequences of strand breaks, cells utilize damage tolerance at the cost of potential mutagenesis. This balance between tolerance and mutagenesis has implications for multiple disease processes and human health. </p>