Determination of G-values for single and double strand break induction in plasmid DNA using agarose gel electrophoresis and a curve-fitting procedure.

Covalently closed circular double-stranded DNA (CC) of native plasmids was used to determine the yield of single strand breaks (ssb) and double strand breaks (dsb) as a consequence of X-irradiation. One ssb transforms DNA of the CC form to the nicked circular form (NC), whereas one dsb produced either directly or from random coincidence of single strand breaks transforms DNA of the CC as well as of the NC form to linear DNA molecules (LI form). Plasmids with more than one dsb are cleaved to linear fragments. DNA (30-800 micrograms/ml) was irradiated in air-saturated sodium phosphate buffer. The different forms of DNA were separated by gel electrophoresis and their amounts measured fluorometrically using ethidium bromide. Large linear DNA fragments with the same electrophoretic mobility as the LI form were considered by using a curve-fitting procedure. From the quantitative changes of each conformation D37 values of ssb and dsb were calculated as a function of the DNA concentration. Finally G-values were calculated by competition plots. The following yields were determined: Gssb 3.4 X 10E-8 molJ-1, and Gdsb 3.3 X 10E-10 molJ-1. Gdsb refers only to those dsb produced directly. Yields are related to strand breaks without further treatment by heat or alkali.

[1]  O. Yamamoto,et al.  Difference in DNA strand break by gamma- and beta-irradiations: an in vitro study. , 1985, Biochemistry international.

[2]  K. Baverstock,et al.  An improved technique of strand break analysis for isodisperse DNA , 1982 .

[3]  N. Oleinick,et al.  The role of DNA damage and repair in the function of eukaryotic genes: radiation-induced single-strand breaks and their rejoining in chromosomal and extrachromosomal ribosomal DNA of tetrahymena. , 1980, Radiation research.

[4]  F. Hutchinson,et al.  Chemical changes induced in DNA by ionizing radiation. , 1985, Progress in nucleic acid research and molecular biology.

[5]  G. P. Schans Gamma-ray induced double-strand breaks in DNA resulting from randomly-inflicted single-strand breaks: temporal local denaturation, a new radiation phenomenon? , 1978 .

[6]  Harold A. Schwarz,et al.  Free Radicals Generated by Radiolysis of Aqueous Solutions. , 1981 .

[7]  B. Zimm,et al.  Theory of gel electrophoresis of DNA , 1985, Biopolymers.

[8]  C. Sonntag,et al.  Oxygen uptake in the radiolysis of aqueous solutions of nucleic acids and their constituents. , 1982, International journal of radiation biology and related studies in physics, chemistry, and medicine.

[9]  R. Lloyd,et al.  Bleomycin-specific fragmentation of double-stranded DNA. , 1978, Biochemistry.

[10]  W. Prütz Inhibition of DNA-ethidium bromide intercalation due to free radical attack upon DNA , 1984, Radiation and environmental biophysics.

[11]  G. P. van der Schans,et al.  Determination of molecular weight distributions of DNA by means of sedimentation in a sucrose gradient. , 1969, Analytical biochemistry.

[12]  F. Studier,et al.  Analysis of bacteriophage T7 early RNAs and proteins on slab gels. , 1973, Journal of molecular biology.

[13]  J. Verberne,et al.  Radiation-induced strand breaks in phi X174 replicative form DNA: an improved experimental and theoretical approach. , 1985, International journal of radiation biology and related studies in physics, chemistry, and medicine.

[14]  J Blok,et al.  The effects of gamma-radiation in DNA. , 1973, Current topics in radiation research quarterly.

[15]  L. Povirk,et al.  DNA double-strand breaks and alkali-labile bonds produced by bleomycin. , 1977, Nucleic acids research.

[16]  P. Serwer,et al.  Conformation of double-stranded DNA during agarose gel electrophoresis: fractionation of linear and circular molecules with molecular weights between 3 X 10(6) and 26 X 10(6). , 1984, Biochemistry.