Thermal inhibition of repair of methylmethane sulfonate-damaged DNA in chick embryo fibroblasts

Chick embryo fibroblasts were treated with the monofunctional alkylating agent methylmethane sulfonate at various concentrations for 1 h at 42°C, rinsed and then incubated post-treatment at various temperatures at which the kinetics of alkali-labile bond disappearance was followed. Growth experiments showed that these cells grew similarly at temperatures of either 37°C or 42°C. Repair as assessed by removal of alkali-labile bond was also similar for postincubation in the temperature range 37–42°C for damage due to methylmethane sulfonate treatment at concentrations less than 1.5 mM. When the postincubation temperature was raised higher than 42.5–43°C, this type of repair was stopped. The normal internal body temperature of adult chickens is about 41.6°C. Hence the present finding indicates that chick cells are much more severely restricted in DNA repair at temperatures above normal than are mammalian cells, which can function in this respect for several deg. C above 37°C.     

Mechanism of maltal hydration catalyzed by beta-amylase: role of protein structure in controlling the steric outcome of reactions catalyzed by a glycosylase.

Crystalline (monomeric) soybean and (tetrameric) sweet potato beta-amylase were shown to catalyze the cis hydration of maltal (alpha-D-glucopyranosyl-2-deoxy-D-arabino-hex-1-enitol) to form beta-2-deoxymaltose. As reported earlier with the sweet potato enzyme, maltal hydration in D2O by soybean beta-amylase was found to exhibit an unusually large solvent deuterium kinetic isotope effect (VH/VD = 6.5), a reaction rate linearly dependent on the mole fraction of deuterium, and 2-deoxy-[2(a)-2H]maltose as product. These results indicate (for each beta-amylase) that protonation is the rate-limiting step in a reaction involving a nearly symmetric one-proton transition state and that maltal is specifically protonated from above the double bond. This is a different stereochemistry than reported for starch hydrolysis. With the hydration catalyzed in H2O and analyzed by gas-liquid chromatography, both sweet potato and soybean beta-amylase were found to convert maltal to the beta-anomer of 2-deoxymaltose. That maltal undergoes cis hydration provides evidence in support of a general-acid-catalyzed, carbonium ion mediated reaction. Of fundamental significance is that beta-amylase protonates maltal from a direction opposite that assumed for protonating starch, yet creates products of the same anomeric configuration from both. Such stereochemical dichotomy argues for the overriding role of protein structures in dictating the steric outcome of reactions catalyzed by a glycosylase, by limiting the approach and orientation of water or other acceptors to the reaction center.     

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.     

Inhibition of yeast respiration and fermentation by benomyl, carbendazim, isocyanates, and other fungicidal chemicals

The inhibition of yeast (Saccharomyces cerevesiae) metabolism by fungicidal chemicals was investigated. Glucose- or ethanol-dependent yeast respiration was measured with an oxygen electrode, and manometric determination of carbon dioxide release was used to measure fermentation. Both respiration and fermentation were inhibited more by benomyl than by identical molar concentrations of its breakdown product, carbendazim. Butyl isocyanate, another benomyl breakdown product, inhibited respiration more but inhibited fermentation less than the parent compound. Of the isocyanates tested, hexyl isocyanate was the most inhibitory towards both activities. Captan was more active and iprodione less active than benomyl. Because benomyl rapidly broke down to carbendazim when it was prepared in 80% ethanol, only 59% of the dissolved benomyl was intact when it was added to yeast to determine its effect on respiration or fermentation.