Search

DNA fragments of defined sequence were used to study DNA strand breakage by gamma radiation in doses ranging from 2,500 to 20,000 rads. The amount of radiation-induced DNA breakage is linearly dose-dependent, and strand scission events occur uniformly at all nucleotide sites, regardless of sequence. The 5'-terminal group at points of breakage is a simple phosphoryl moiety. Two types of radiation-induced 3' termini were identified. One species of 3' terminus is a simple phosphoryl group. The other species of 3' terminus is neither a hydroxyl nor a phosphoryl group. A model for radiation-induced DNA strand scission that involves destruction of the deoxyribose moiety is presented. Similarities among DNA strand scission events created by gamma radiation, ferrous ion, doxorubicin, and bleomycin are discussed.

gamma-Irradiation of DNA in vitro produces two types of single strand breaks. Both types of strand breaks contain 5'-phosphate DNA termini. Some strand breaks contain 3'-phosphate termini, some contain 3'-phosphoglycolate termini (Henner, W.D., Rodriguez, L.O., Hecht, S. M., and Haseltine, W. A. (1983) J. Biol. Chem. 258, 711-713). We have studied the ability of prokaryotic enzymes of DNA metabolism to act at each of these types of gamma-ray-induced 3' termini in DNA. Neither strand breaks that terminate with 3'-phosphate nor 3'-phosphoglycolate are substrates for direct ligation by T4 DNA ligase. Neither type of gamma-ray-induced 3' terminus can be used as a primer for DNA synthesis by either Escherichia coli DNA polymerase or T4 DNA polymerase. The 3'-phosphatase activity of T4 polynucleotide kinase can convert gamma-ray-induced 3'-phosphate but not 3'-phosphoglycolate termini to 3'-hydroxyl termini that can then serve as primers for DNA polymerase. E. coli alkaline phosphatase is also unable to hydrolyze 3'-phosphoglycolate groups. The 3'-5' exonuclease actions of E. coli DNA polymerase I and T4 DNA polymerase do not degrade DNA strands that have either type of gamma-ray-induced 3' terminus. E. coli exonuclease III can hydrolyze DNA with gamma-ray-induced 3'-phosphate or 3'-phosphoglycolate termini or with DNase I-induced 3'-hydroxyl termini. The initial action of exonuclease III at 3' termini of ionizing radiation-induced DNA fragments is to remove the 3' terminal phosphate or phosphoglycolate to yield a fragment of the same nucleotide length that has a 3'-hydroxyl terminus. These results suggest that repair of ionizing radiation-induced strand breaks may proceed via the sequential action of exonuclease, DNA polymerase, and DNA ligase. The possible role of exonuclease III in repair of gamma-radiation-induced strand breaks is discussed.

The ratio of alkali-labile lesions to total DNA adducts for DNA modified by an active metabolite of benzo(a)pyrene was investigated using DNA sequencing methodology. About 40% of the adducts formed result in alkali-labile sites. About 25% of the lesions were alkali-labile at positions of guanine, 10% at adenine, and 5% at cytosine. This study highlights the potential role of adducts other than the N2-substituted guanine in mutagenic and carcinogenic effects of benzo(a)pyrene.

Ultraviolet light induces alkaline labile lesions in DNA. These lesions occur at the bipyrimidine sites T-C, C-C, and T-T, and do not result from the formation of pyrimidine cyclobutane dimers. To examine the chemical nature of the alkaline labile lesions, pyrimidine dinucleotides (2'-deoxythymidylyl-(3' leads to 5')-2'-deoxycytidine, 2'-deoxythymidylyl-(3' leads to 5')-2'-deoxythymidine, 2'-deoxycytidylyl-(3' leads to 5')-2-deoxycytidine, and 2'-deoxycytidylyl-(3' leads to 5')-2'-deoxythymidine) were used as a model system. Ultraviolet light irradiation of all four dinucleotides resulted in the formation of substantial quantities (relative to cyclobutane pyrimidine dimers) of products that were red-shifted in UV absorbance and fluorescent. These products are identified as precursors to the 6-4'-[pyrimidin-2'-one]-pyrimidine class of products that have been previously shown to occur in UV-irradiated DNA. The fluorescent products were found to be alkaline labile; the products contained a 3'-phosphate end group after alkali treatment. Two of the fluorescent products have been found in enzymatic digests of high dose UV-irradiated salmon sperm DNA, the T-C and T-T UV-induced products. The relative rates of formation of these products in 32P-radiolabeled dinucleotides were measured. We conclude that the alkaline labile lesions observed in DNA at the bipyrimidine sites are the same as those that yield the 6-4 products upon acid hydrolysis of DNA. The mechanisms for the formation and the consequences of DNA structure for these lesions as well as the possible biological significance of this class of UV damage are discussed.

The effect of purified Escherichia coli DNA photolyase on the UV light-induced pyrimidine-pyrimidone (6-4) photoproduct and cyclobutane pyrimidine dimer was investigated in vitro using enzyme purified from cells carrying the cloned phr gene (map position, 15.7 min). Photoproducts were examined both as site-specific lesions in end-labeled DNA and as chromatographically identified products in uniformly labeled DNA. E. coli DNA photolyase removed cyclobutane dimers but had no activity on pyrimidine-pyrimidone (6-4) photoproducts. Photoreactivation can therefore be used to separate the biological effects of these two UV light-induced molecular lesions.

A single locus (designated c-fes) in the human genome which exhibits homology to the transformation-specific onc gene (v-fes) of Snyder-Theilen feline sarcoma virus was identified by the Southern blot technique. Recombinant clones containing 16- to 18-kilobase inserts of human DNA including the c-fes locus were constructed. Restriction endonuclease mapping of these clones verified their identity with native human c-fes and demonstrated the presence of at least two sequences in human c-fes interrupting v-fes-homologous regions. The v-fes-homologous locus in the human genome spans about 4 kilobases. The 5'-3' orientation of the c-fes clones with respect to feline sarcoma virus proviral DNA was determined. The region of the human genome that is homologous to v-fes is proximal to the highly reiterated human Alu sequence but not to the highly reiterated human alphoid sequence.