Inhibitory effect of conjugated eicosapentaenoic acid on human DNA topoisomerases I and II

DNA topoisomerases (topos) and DNA polymerases (pols) are involved in many aspects of DNA metabolism such as replication reactions. We reported previously that long chain unsaturated fatty acids such as polyunsaturated fatty acids (PUFA) (i.e., eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA)) inhibited the activities of eukaryotic pols in vitro. In the present study, we found that PUFA also inhibited human topos I and II activities, and the inhibitory effect of conjugated fatty acids converted from EPA and DHA (cEPA and cDHA) on pols and topos was stronger than that of normal EPA and DHA. cEPA and cDHA inhibited the activities of mammalian pols and human topos, but did not affect the activities of plant and prokaryotic pols or other DNA metabolic enzymes tested. cEPA was a stronger inhibitor than cDHA with IC(50) values for mammalian pols and human topos of 11.0-31.8 and 0.5-2.5 microM, respectively. Therefore, the inhibitory effect of cEPA on topos was stronger than that on pols. Preincubation analysis suggested that cEPA directly bound both topos I and II, but did not bind or interact with substrate DNA. This is the first report that conjugated PUFA such as cEPA act as inhibitors of pols and topos. The results support the therapeutic potential of cEPA as a leading anti-cancer compound that poisons pols and topos.     

Resin probe analysis. II. Amino acid coupling reactions.

Resin probe analysis has been employed to evaluate the availability of dicyclohexylcarbodiimide (DCC)-activated amino acids, the relationship between coupling time and reaction yield, and the influence of triethylamine (TEA) concentration on peptide bond formation. Results are presented for five amino acids which indicate that the coupling reactions plateau within 5 min, and no significant increase in yield is observed for longer incubation times. Large decreases in coupling yield (70–90%) were observed at concentrations of TEA above 0.01 m. Inactivation appears to be dependent in part upon amino acid structural features. In the absence of TEA, DCC-activated t-butyloxycarbonyl (Boc)-glycine was stable in the activated state for hours. peptide bond formation showed little or no amino acid concentration-dependence in the range of 0.01–0.04 m. Resin probe experiments provide quantitative data on reaction progress and factors that influence the availability and reactivity of activated amino acids.