Regioselective degradation of [beta] 1,3 glucan by ferrous ion and hydrogen peroxide (Fenton oxidation).

Many species use Fe+2 and H2O2 to oxidize a wide variety of compounds to simpler molecules. Both pathogen killing by leukocytes (neutrophils and lymphocytes) and degradation of cellulose by brown rot fungi rely on excretion of Fe+2 ions and H2O2, the Fenton reagent. To elucidate the mechanism of Fenton oxidation of carbohydrates, ß1,3 glucan (laminaran), a major fungal wall polysaccharide, was oxidized using a molar ratio of monomer/Fe+2/H2O2 of 10:1:1 (primarily). We labeled the reaction products and profiled them as fluorescent-labeled molecules in polyacrylamide gels and as hydrophobic-tagged molecules using reverse phase liquid chromatography/mass spectrometry (HPLC/MS). Sub-stoichiometric concentrations of Fe+2 and H2O2 fragmented laminaran into smaller molecules containing carbonyl and carboxylic acid groups visible on fluorescent-labeled carbohydrate polyacrylamide gel electrophoresis. HPLC/MS analysis of glucan fragments showed masses consistent with six classes of molecules: aldoses, dialdoses, uronic acids, hexosuloses, aldonic acids, and hexulosonic acids. The results were consistent with published mechanisms where hydrogen radical (H•) abstraction from a C-H or O-H bond begins a cascade of reactions leading to 1) C-C bond cleavage to produce aldose/dialdose pairs; 2) oxo-group (O = ) addition to produce uronic and aldonic acids; 3) hydroxyl group (HO-) addition to produce gluconolactone and hexosuloses; and 4) hexulosonic acids. Most products resulted from regioselective H• abstractions characteristic of oxidations by ferryl-oxo ion [(FeO)+2] or perferryl-oxo ion [(FeO)+3] in close contact with specific positions in the glycan. Therefore, oxidations initiated by regioselectively-bound Fe ions were the predominant initiators of polysaccharide degradations.

A Protein in the Yeast Saccharomyces cerevisiae Presents DNA Binding Homology to the p53 Checkpoint Protein and Tumor Suppressor.

Saccharomyces cerevisiae does not contain a p53 homolog. Utilizing this yeast as an in vivo test tube model, our aim was to investigate if a yeast protein would show p53 DNA binding homology. Electrophoretic mobility shift analyses revealed the formation of specific DNA-protein complexes consisting of S. cerevisiae nuclear protein(s) and oligonucleotides containing p53 DNA binding sites. A S. cerevisiae p53 binding site factor (Scp53BSF) bound to a p53 synthetic DNA-consensus sequence (SCS) and a p53 binding-site sequence from the MDM2 oncogene. The complexes were of comparable size. Like mammalian p53, the affinity of Scp53BSF for the SCS oligonucleotide was higher than for the MDM2 oligonucleotide. Binding of Scp53BSF to the SCS and MDM2 oligonucleotides was strongly competed by unlabeled oligonucleotides containing mammalian p53 sites, but very little by a mutated site oligonucleotide. Importantly, Scp53BSF-DNA binding activity was significantly induced in extracts from cells with DNA damage. This resulted in dose-dependent coordinated activation of transcription when using p53-binding site reporter constructs. An ancient p53-like DNA binding protein may have been found, and activation of DNA-associated factors to p53 response elements may have functions not yet determined.

Impaired mitochondrial function protects against free radical-mediated cell death.

Free radical damage can have fatal consequences. Mitochondria carry out essential cellular functions and produce high levels of reactive oxygen species (ROS). Many agents also generate ROS. Using the yeast Saccharomyces cerevisiae as a eukaryotic model, the role of functional mitochondria in surviving free radical damage was investigated. Respiratory-deficient cells lacking mitochondrial DNA (rho(0)) were up to 100-fold more resistant than isogenic rho(+) cells to killing by ROS generated by the bleomycin-phleomycin family of oxidative agents. Up to approximately 90% of the survivors of high oxidative stress lost mitochondrial function and became "petites." The selective advantage of respiratory deficiency was studied in several strains, including DNA repair-deficient rad52/rad52 and blm5/blm5 diploid strains. These mutant strains are hypersensitive to lethal effects of free radicals and accumulate more DNA damage than related wild-type strains. Losses in mitochondrial function were dose-dependent, and mutational alteration of the RAD52 or BLM5 gene did not affect the resistance of surviving cells lacking mitochondrial function. The results indicate that inactivation of mitochondrial function protects cells against lethal effects of oxygen free radicals.

Loss of a 20S proteasome activator in Saccharomyces cerevisiae downregulates genes important for genomic integrity, increases DNA damage, and selectively sensitizes cells to agents with diverse mechanisms of action.

Cytoprotective functions of a 20S proteasome activator were investigated. Saccharomyces cerevisiae Blm10 and human 20S proteasome activator 200 (PA200) are homologs. Comparative genome-wide analyses of untreated diploid cells lacking Blm10 and growing at steady state at defined growth rates revealed downregulation of numerous genes required for accurate chromosome structure, assembly and repair, and upregulation of a specific subset of genes encoding protein-folding chaperones. Blm10 loss or truncation of the Ubp3/Blm3 deubiquitinating enzyme caused massive chromosomal damage and cell death in homozygous diploids after phleomycin treatments, indicating that Blm10 and Ubp3/Blm3 function to stabilize the genome and protect against cell death. Diploids lacking Blm10 also were sensitized to doxorubicin, hydroxyurea, 5-fluorouracil, rapamycin, hydrogen peroxide, methyl methanesulfonate, and calcofluor. Fluorescently tagged Blm10 localized in nuclei, with enhanced fluorescence after DNA replication. After DNA damage that caused a classic G2/M arrest, fluorescence remained diffuse, with evidence of nuclear fragmentation in some cells. Protective functions of Blm10 did not require the carboxyl-terminal region that makes close contact with 20S proteasomes, indicating that protection does not require this contact or the truncated Blm10 can interact with the proteasome apart from this region. Without its carboxyl-terminus, Blm10((-339aa)) localized to nuclei in untreated, nonproliferating (G(0)) cells, but not during G(1) S, G(2), and M. The results indicate Blm10 functions in protective mechanisms that include the machinery that assures proper assembly of chromosomes. These essential guardian functions have implications for ubiquitin-independent targeting in anticancer therapy. Targeting Blm10/PA200 together with one or more of the upregulated chaperones or a conventional treatment could be efficacious.

Expression of the expanded YFL007w ORF and assignment of the gene name BLM10.

This report assigns a gene name to an extended ORF in Saccharomyces cerevisiae, and provides the first evidence from Northern analyses that the ORF is expressed and the transcript is the predicted size.