Induced Pyrimidine Dimers Research Articles

The fate of pyrimidine dimers in ultraviolet-irradiated Chlamydomonas.

Abstract— We have developed a chromatographic technique for the separation of 32P‐labeled pyrimidine nucleotide dimers of the form PypPy from 32P‐phosphate in enzymatic hydroly sates of ultraviolet‐irradiated DNA. Application of this technique to 32P‐labeled Chlamydomonas reinhardii shows that ultraviolet irradiation of this organism induces pyrimidine dimers in both nuclear and chloroplast DNA. We have found no evidence that these dimers are excised from either DNA species after several hours incubation under non‐photoreactivating conditions. A function has been derived to permit the pyrimidine‐dimer content determined from radioactive‐thymine‐labeled cells to be conveniently compared to that obtained from 32P‐phosphate‐labeled cells.

A comparative study of the effects of UV irradiation upon diploid cultures of yeast defective at therad 3 locus

Therad 3 gene ofSaccharomyces cerevisiae appears to code for one of the enzymes involved in the repair of UV induced pyrimidine dimers. Haploid and diploid yeast cultures carrying different mutant alleles of therad 3 gene show considerable variation in their responses to both UV inactivation and post UV modifying treatments such as liquid holding in basal medium and photoreactivation. Positive liquid holding recovery was shown only by those diploid cultures containing alleles which conferred the highest levels of UV resistance. The results indicate that liquid holding recovery in yeast requires the activity of the excision-repair pathway for expression.

Induction of pyrimidine dimers in bacterial DNA by 365 nm radiation.

Induction of pyrimidine dimers in bacterial DNA by 365 nm radiation.

Exchanges between DNA strands in ultraviolet-irradiated Escherichia coli

Ultraviolet induced pyrimidine dimers remain in the DNA of excision-defective mutants of Escherichia coli for at least several hours after irradiation. If kept in the dark, the survival of colony-forming ability of these mutant bacteria depends upon recombinational repair, a mechanism which is controlled by the gene recA. In u.v.-irradiated bacteria, newly synthesized DNA strands are of low molecular weight, but these strands are subsequently joined into high molecular weight chains, as is shown by sedimenting the bacterial DNA in alkaline sucrose gradients. These longer chains may contribute to cell survival by acting as templates for the synthesis of high molecular weight strands during further replication. At times greater than 90 minutes after irradiation, the molecular weight of newly synthesized DNA covers a wide range and extends fully to the maximum found with unirradiated cells, thus indicating the presence of high molecular weight templates virtually free from damaged bases. Evidently the u.v. photoproducts tend to remain in the original strands where they were formed, rather than becoming distributed equally among all the strands with each subsequent replication. Recombinational repair may depend upon genetic exchanges between sister duplexes. To see whether such exchanges could be detected after u.v.-irradiation, cells were grown for several generations in medium containing 13C and 15N. They were then exposed to various doses of ultraviolet light and grown for less than a generation in light medium containing [ 3H]thymidine, so that part of their DNA was hybrid in density, with the 3H label in the light strand. Under these conditions, light 3H-labeled chains will become linked to heavy strands if exchanges occur between sister duplexes. The DNA was extracted from unirradiated cells, heat denatured and centrifuged in neutral cesium chloride gradients, and was found to separate into heavy and light bands. Denatured DNA from u.v.-irradiated cells did not separate completely, but contained strands of intermediate density which separated into heavy and light components only after shearing to 5 × 10 5 molecular weight, thus indicating that segments of molecular weight greater than 5 × 10 5 had been exchanged. To judge from the fraction of the 3H-labeled molecules that were of intermediate density, one exchange occurred for every one to two pyrimidine dimers in the DNA replicated in the 3H-labeled medium. Sister exchanges involving a single strand of each duplex, may insert the correct bases sequence into the gap opposite each pyrimidine dimer, and thus promote the reconstruction of a genome with the complete base sequence needed for the survival of colony-forming ability.

Isolation of ultraviolet light induced pyrimidine dimers from enzymic hydrolyzates of DNA

A method has been developed for the isolation of ultraviolet light induced pyrimidine dimers from enzymic hydrolyzates of DNA. The dimers are isolated in the form ▪, thus making it possible to use 32P-labeled DNA for the study of ultraviolet light induced pyrimidine dimers.

Dark reactivation of damage induced by ultraviolet light in mammalian cells in vitro.

INDUCTION of pyrimidine dimers in cellular DNA by irradiation with ultraviolet light and the molecular processes involved in the repair of this damage (photoreactivation1 and dark reactivation2–3) are established in bacteria, bacteriophage and transforming DNA. Investigations of the reactivation of ultraviolet-induced damage have been extended from micro-organisms to cover cultured mammalian cell lines4–6.

Normal Replication of DNA after Repair Replication in Bacteria

AN early step in the dark repair of ultra-violet damaged DNA in bacteria is the excision of the damaged single strand regions1,2. It has been postulated that a later step involves the replacement of the excised segments with undamaged deoxyribonucleotides, utilizing the complementary base pairing with the intact single strand1,2. Direct physical evidence for this repolymerization step has been obtained in our laboratory employing the procedure of DNA density labelling with 5-bromouracil and density distribution analysis in caesium chloride density gradients3 to distinguish semiconservative replication from the non-conservative mode of repair replication4. This non-conservative replication has been observed in bacteria after treatment with ultra-violet4, nitrogen mustard5, nitrosoguanidine (unpublished work of E. Cerda-Olmedo and P. C. Hanawalt), or thymine starvation6. The belief that this is the postulated repair synthesis step is strengthened by the results of a number of control experiments as follows. Non-conservative DNA replication is not observed in bacteria in which ultra-violet induced pyrimidine dimers have been repaired in situ by photo-reactivating conditions before density labelling4. It is not observed after ultra-violet irradiation of a strain, E. coli BS–1, sensitive to ultra-violet light which is unable to perform the excision step in the repair sequence7. Non-conservative replication has also been demonstrated by the use of deuterium oxide and nitrogen-15 as density labels to prevent possible artefacts caused by the pathogenicity of 5-bromouracil8. We report here an additional control experiment which demonstrates that the DNA molecules damaged by ultra-violet irradiation which have undergone non-conservative replication are then capable of normal semiconservative replication.