Inefficient bypass of an abasic site by DNA polymerase eta

DNA polymerase eta (Pol eta) bypasses a cis-syn thymine-thymine dimer efficiently and accurately, and inactivation of Pol eta in humans results in the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Also, Pol eta bypasses the 8-oxoguanine lesion efficiently by predominantly inserting a C opposite this lesion, and it bypasses the O(6)-methylguanine lesion by inserting a C or a T. To further assess the range of DNA lesions tolerated by Pol eta, here we examine the bypass of an abasic site, a prototypical noninstructional lesion. Steady-state kinetic analyses show that both yeast and human Pol eta are very inefficient in both inserting a nucleotide opposite an abasic site and in extending from the nucleotide inserted. Hence, Pol eta bypasses this lesion extremely poorly. These results suggest that Pol eta requires the presence of template bases opposite both the incoming nucleotide and the primer terminus to catalyze efficient nucleotide incorporation.     

Fidelity and damage bypass ability of Schizosaccharomyces pombe Eso1 protein, comprised of DNA polymerase eta and sister chromatid cohesion protein Ctf7

DNA polymerase eta (Poleta) functions in error-free bypass of ultraviolet light-induced DNA lesions, and mutational inactivation of Poleta in humans causes the cancer prone syndrome, the variant form of xeroderma pigmentosum (XPV). Both Saccharomyces cerevisiae and human Poleta efficiently insert two adenines opposite the two thymines of a cyclobutane pyrimidine dimer. Interestingly, in the fission yeast Schizosaccharomyces pombe, the eso1(+) encoded protein is comprised of two domains, wherein the NH(2) terminus is highly homologous to Poleta, and the COOH terminus is highly homologous to the S. cerevisiae Ctf7 protein which is essential for the establishment of sister chromatid cohesion during S phase. Here we characterize the DNA polymerase activity of S. pombe GST-Eso1 fusion protein and a truncated version containing only the Poleta domain. Both proteins exhibit a similar DNA polymerase activity with a low processivity, and steady-state kinetic analyses show that on undamaged DNA, both proteins misincorporate nucleotides with frequencies of approximately 10(-2) to 10(-3). We also examine the two proteins for their ability to replicate a cyclobutane pyrimidine dimer-containing DNA template and find that both proteins replicate through the lesion equally well. Thus, fusion with Ctf7 has no significant effect on the DNA replication or damage bypass properties of Poleta. The possible role of Ctf7 fusion with Poleta in the replication of Cohesin-bound DNA sequences is discussed.     

Fidelity and processivity of Saccharomyces cerevisiae DNA polymerase eta

The yeast RAD30 gene functions in error-free replication of UV-damaged DNA, and RAD30 encodes a DNA polymerase, pol eta, that has the ability to efficiently and correctly replicate past a cis-syn-thymine-thymine dimer in template DNA. To better understand the role of pol eta in damage bypass, we examined its fidelity and processivity on nondamaged DNA templates. Steady-state kinetic analyses of deoxynucleotide incorporation indicate that pol eta has a low fidelity, misincorporating deoxynucleotides with a frequency of about 10(-2) to 10(-3). Also pol eta has a low processivity, incorporating only a few nucleotides before dissociating. We suggest that pol eta's low fidelity reflects a flexibility in its active site rendering it more tolerant of DNA damage, while its low processivity limits its activity to reduce errors.     

Relationships among seizures, extracellular amino acid changes, and neurodegeneration induced by 4-aminopyridine in rat hippocampus: a microdialysis and electroencephalographic study

4-Aminopyridine is a powerful convulsant that induces the release of neurotransmitters, including glutamate. We report the effect of intrahippocampal administration of 4-aminopyridine at six different concentrations through microdialysis probes on EEG activity and on concentrations of extracellular amino acids and correlate this effect with histological changes in the hippocampus. 4-Aminopyridine induced in a concentration-dependent manner intense and frequent epileptic discharges in both the hippocampus and the cerebral cortex. The three highest concentrations used induced also a dose-dependent enhancement of extracellular glutamate, aspartate, and GABA levels and profound hippocampal damage. Neurodegenerative changes occurred in CA1, CA3, and CA4 subfields, whereas CA2 was spared. In contrast, microdialysis administration of a depolarizing K+ concentration and of tetraethylammonium resulted in increased amino acid levels but no epileptic activity and no or moderate neuronal damage. These results suggest that seizure activity induced by 4-aminopyridine is due to a combined action of excitatory amino acid release and direct stimulation of neuronal firing, whereas neuronal death is related to the increased glutamate release but is independent of seizure activity. In addition, it is concluded that the glutamate release-inducing effect of 4-aminopyridine results in excitotoxicity because it occurs at the level of nerve endings, thus permitting the interaction of glutamate with its postsynaptic receptors, which is probably not the case after K+ depolarization.     

Angular variances for internal bond rotations of side chains in GXG-based tripeptides derived from (13)C-NMR relaxation measurements: Implications to protein folding

The study of backbone and side-chain internal motions in proteins and peptides is crucial to having a better understanding of protein/peptide "structure" and to characterizing unfolded and partially folded states of proteins and peptides. To achieve this, however, requires establishing a baseline for internal motions and motional restrictions for all residues in the fully, solvent-exposed "unfolded state." GXG-based tripeptides are the simpliest peptides where residue X is fully solvent exposed in the context of an actual peptide. In this study, a series of GXG-based tripeptides has been synthesized with X being varied to include all twenty common amino acid residues. Proton-coupled and -decoupled (13)C-nmr relaxation measurements have been performed on these twenty tripeptides and various motional models (Lipari-Szabo model free approach, rotational anisotropic diffusion, rotational fluctuations within a potential well, rotational jump model) have been used to analyze relaxation data for derivation of angular variances and motional correlation times for backbone and side-chain chi(1) and chi(2) bonds and methyl group rotations. At 298 K, backbone motional correlation times range from about 50 to 85 ps, whereas side-chain motional correlation times show a much broader spread from about 18 to 80 ps. Angular variances for backbone phi,psi bond rotations range from 11 degrees to 23 degrees and those for side chains vary from 5 degrees to 24 degrees for chi(1) bond rotations and from 5 degrees to 27 degrees for chi(2) bond rotations. Even in these peptide models of the "unfolded state," side-chain angular variances can be as restricted as those for backbone and beta-branched (valine, threonine, and isoleucine) and aromatic side chains display the most restricted motions probably due to steric hinderence with backbone atoms. Comparison with motional data on residues in partially folded, beta-sheet-forming peptides indicates that side-chain motions of at least hydrophobic residues are less restricted in the partially folded state, suggesting that an increase in side-chain conformational entropy may help drive early-stage protein folding. Copyright 1999 John Wiley & Sons, Inc.     

Efficient and error-free replication past a minor-groove DNA adduct by the sequential action of human DNA polymerases iota and kappa

DNA polymerase iota (Poliota) is a member of the Y family of DNA polymerases, which promote replication through DNA lesions. The role of Poliota in lesion bypass, however, has remained unclear. Poliota is highly unusual in that it incorporates nucleotides opposite different template bases with very different efficiencies and fidelities. Since interactions of DNA polymerases with the DNA minor groove provide for the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, we considered the possibility that Poliota differs from other DNA polymerases in not being as sensitive to distortions of the minor groove at the site of the incipient base pair and that this enables it to incorporate nucleotides opposite highly distorting minor-groove DNA adducts. To check the validity of this idea, we examined whether Poliota could incorporate nucleotides opposite the gamma-HOPdG adduct, which is formed from an initial reaction of acrolein with the N(2) of guanine. We show here that Poliota incorporates a C opposite this adduct with nearly the same efficiency as it does opposite a nonadducted template G residue. The subsequent extension step, however, is performed by Polkappa, which efficiently extends from the C incorporated opposite the adduct. Based upon these observations, we suggest that an important biological role of Poliota and Polkappa is to act sequentially to carry out the efficient and accurate bypass of highly distorting minor-groove DNA adducts of the purine bases.     

Progressive iron accumulation induces a biphasic change in the glutathione content of neuroblastoma cells

Glutathione (GSH) constitutes the single most important antioxidant in neurons, whereas iron causes oxidative stress that leads to cell damage and death. Although GSH and iron produce opposite effects on redox cell status, no mechanistic relationships between iron and GSH metabolism are known. In this work, we evaluated in SH-SY5Y neuroblastoma cells the effects of iron accumulation on intracellular GSH metabolism. After 2 d exposure to increasing concentrations of iron, cells underwent concentration-dependent iron accumulation and a biphasic change in intracellular GSH levels. Increasing iron from 1 to 5 microM resulted in a marked increase in intracellular oxidative stress and increased GSH levels. Increased GSH levels were due to increased synthesis. Further increases in iron concentration led to significant reduction in both reduced (GSH) and total (GSH + (2 x GSSG)) glutathione. Cell exposure to high iron concentrations (20-80 microM) was associated with a marked decrease in the GSH/GSSG molar ratio and the GSH half-cell reduction potential. Moreover, increasing iron from 40 to 80 microM resulted in loss of cell viability. Iron loading did not change GSH reductase activity but induced significant increases in GSH peroxidase and GSH transferase activities. The changes in GSH homeostasis reported here recapitulate several of those observed in Parkinson's disease substantia nigra. These results support a model by which progressive iron accumulation leads to a progressive decrease in GSH content and cell reduction potential, which finally results in impaired cell integrity.     

Synthesis of dihydronaphthofurandiones and dihydrofuroquinolinediones with trypanocidal activity and analysis of their stereoelectronic properties

The synthesis of dihydronaphthofurandione and dihydrofuroquinolinedione derivatives 4-11 was performed through Diels-Alder reactions of dihydrobenzofurandione 1 with several carbodienes and acrolein N,N-dimethylhydrazone. Then, the use of 5-bromobenzofurandione 2 toward 1,3-pentadiene and the 1-azadiene afforded quinones 6 and 11 with a total regioselectivity. All the prepared quinones were tested for trypanocidal activity in vitro against Trypanosoma epimastigotes, Tulahuen strain. Among the tested compounds, the furoquinolinediones 10 and 11 have shown potent trypanocidal activities but, only the 1,5-regioisomer (11) was found active as a redox cycling agent. Calculation of their stereoelectronic properties by the density-functional theory method provided a new insight for the trypanocidal activity of these heterocyclic quinones.