One-electron oxidation of plasmid DNA by selenium(V) species.
OBJECTIVE
To employ the gamma-radiation-generated selenium(V) one-electron-oxidizing agent SeO3*- for the preparation of guanyl radicals in plasmid DNA, and to compare the behaviour of this reagent with that of other similarly reactive oxidant species.


METHODS
Plasmid DNA in aerobic aqueous solution was irradiated with 137Cs gamma-rays (662 keV). The solutions also contained up to 4x10(-2) mol x dm(-3) sodium selenate (Na2SeO4) and/or up to 10(-1) mol x dm(-3) sodium biselenite (NaHSeO3), as well as auxiliary scavengers such as DMSO or glycerol. In some cases, reducing agents such as ferrocyanide were also present. After irradiation, the plasmid was incubated with the Escherichia coli base excision-repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). These treatments produced strand breaks in the plasmid. The yields of these strand breaks were quantified by agarose gel electrophoresis.


RESULTS
In general, gamma-irradiation produced single-strand breaks (SSB) in plasmid DNA. Subsequent incubation with the endonuclease FPG increased the SSB yield by a factor of 2-100-fold. The smallest effects of FPG were observed when only DMSO or glycerol were present during irradiation. FPG incubation produced significantly larger increases in the SSB yield after gamma-irradiation in the additional presence of selenate and/or biselenite. The largest effect of FPG was observed after gamma-irradiation in the presence of 10(-2) mol x dm(-3) sodium selenate and 10(-1) mol x dm(-3) glycerol. This was indicative of extensive oxidative damage to the plasmid under these conditions and provided evidence for guanine oxidation mediated by SeO3*-. The large effect of FPG was strongly attenuated by the addition of reducing agents such as ferrocyanide. The observations suggest that these reducing agents exert their effects through the reduction of an intermediate guanyl radical.


CONCLUSIONS
By comparing the yields of breaks produced after gamma-irradiation under a range of conditions, it is possible to formulate a reaction scheme that describes the chemical reactions responsible for the formation of strand breaks and FPG-sensitive sites. By applying this scheme to the data, we can quantify rate constants for the reduction of DNA guanyl radicals by reducing agents. This reaction is of particular interest to radiation biology because it is the equivalent of the repair of DNA damage by the direct effect of ionizing radiation.
Milligan
J R
JR
Department of Radiology, University of California at San Diego, La Jolla, CA 92093, USA. jmilligan@ucsd.edu
Aguilera
J A
JA
Paglinawan
R A
RA
Ward
J F
JF
D001665:Binding Sites; D004249:DNA Damage; D004260:DNA Repair; D043382:DNA-Formamidopyrimidine Glycosylase; D004307:Dose-Response Relationship, Radiation; D004583:Electrons; D029968:Escherichia coli Proteins; D005295:Ferrocyanides / Q000494:pharmacology; D005720:Gamma Rays; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D010084:Oxidation-Reduction; D010957:Plasmids / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects*; D011853:Radiobiology; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000528:radiation effects; D064586:Selenic Acid; D012643:Selenium / Q000737:chemistry / Q000494:pharmacology* / Q000528:radiation effects; D018036:Selenium Compounds / Q000737:chemistry / Q000494:pharmacology / Q000528:radiation effects; D018038:Sodium Selenite / Q000737:chemistry / Q000494:pharmacology / Q000528:radiation effects
D029968:Escherichia coli Proteins; D005295:Ferrocyanides; D017382:Reactive Oxygen Species; D018036:Selenium Compounds; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; C020354:hexacyanoferrate II; D012643:Selenium; D018038:Sodium Selenite; D064586:Selenic Acid