Utilization of salmon milt DNA against UV damage.

We examined the effect of ultraviolet (UV) irradiation on the UV spectra and radical scavenging activity of DNA strands and found that the absorption spectra of salmon milt DNA was extended up to about 350 nm after ultraviolet C (UVC, 100-280 nm) irradiation with 300 kJ/m(2). The UV B (UVB, 280-315 nm) protection ability of UVC-irradiated salmon milt DNA for a single-stranded target DNA (19-mer) was further studied. The percentage of damaged target DNA after 50 kJ/m(2) of UVB irradiation in the presence of UVC-irradiated salmon milt DNA, UVC-unirradiated salmon milt DNA, and 2-phenylbenzimidazole sulfonic acid was estimated to be 24.6%, 27.0%, and 18.9%, respectively. Moreover, the ultraviolet A (UVA, 315-400 nm)/UVB ratio and critical wavelength of natural (UVC-unirradiated) salmon milt DNA were estimated to be 0.13 and 313 nm, respectively, whereas those of the UVC-irradiated salmon milt DNA were 0.34 and 375 nm, respectively. Interestingly, the value of 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity in UVC-irradiated salmon milt DNA was about five times higher than that of UVC-unirradiated salmon milt DNA. These results indicate that the UVC-irradiated salmon milt DNA could be useful as a protector against a wide range of UV light from UVC approximately UVA.

Regulation of DNA nucleases by molecular crowding.

Here, we examined the effects of molecular crowding on the function, structure and stability of nucleases. We found that the hydrolysis of a 29-mer double-stranded DNA by the endonucleases DNase I and S1 nuclease was substantially enhanced by molecular crowding using polyethylene glycol (PEG); however, molecular crowding had little effect on hydrolysis by exo III and exo I exonucleases. Moreover, kinetic analysis showed that the maximum velocity for the reaction of DNase I at 25 degrees C was increased from 0.1 to 2.7 microM/min by molecular crowding with 20% (w/v) PEG, whereas that of exonuclease I at 37 degrees C decreased from 2.2 to 0.4 microM/min. In contrast, molecular crowding did not significantly affect the Michaelis constant of DNase I or exonuclease I. These results indicate that molecular crowding has different effects on the catalytic activities of exonucleases and endonucleases.

Effect of molecular crowding on DNA polymerase activity.

Live cells contain high concentrations of macromolecules, but almost all experimental biochemical data have been generated from dilute solutions that do not reflect conditions in vivo. To understand biomolecular behavior in vivo, properties studied in vitro are extrapolated to conditions in vivo; however, the molecular conditions within live cells are inherently crowded. The present study investigates the effect of molecular crowding on DNA polymerase activity using polyethylene glycol PEG of various molecular weights as a crowding agent. Polymerase activity assays under various conditions demonstrated that the activities of T7 and Taq DNA polymerases depend on the molecular weight and concentration of the crowding agent. Furthermore, equilibrium and kinetic analyses demonstrated that the binding affinity and catalytic activity of the polymerase increase and decrease, respectively, with increasing PEG concentrations. Based on quantitative parameters of the polymerase reactions, we improved the efficiency of PCR amplification under conditions of molecular crowding. These results suggest that quantitative measurements of biomolecular structure and function are useful for understanding the behavior of biomolecules in vivo and for biotechnology applications in vitro.

Lactobacillus reuteri ATCC 53608 mdh gene cloning and recombinant mannitol dehydrogenase characterization.

A gene encoding mannitol-2-dehydrogenase (E.C. 1.1.1.138) (MDH) was cloned from Lactobacillus reuteri and expressed in Escherichia coli. The 1,008-bp gene encodes a protein consisting of 336 amino acids, with a predicted molecular mass of 35,920 Da. The deduced amino acid sequence of L. reuteri MDH (LRMDH) is 77% and 76% similar to the MDHs from Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, respectively. The purified recombinant enzyme appears as a single band of 40 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but gel filtration indicates that the native enzyme is a dimer. The optimum temperature for the recombinant enzyme is 37 degrees C, the pH optima for D-fructose reduction and D-mannitol oxidation are 5.4 and 6.2, respectively. The K(m) values for NAD (9 mM) and NADH (0.24 mM) are significantly higher than those for NADP (0.35 mM) and NADPH (0.04 mM). The K(m) values of LRMDH for D-fructose and D-mannitol are 34 mM and 54 mM, respectively. Contrary to what the enzyme sequence suggests, recombinant LRMDH contains a single catalytic zinc per subunit.