UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells.

Abstract

We used a simian virus 40-based shuttle vector plasmid, pZ189, to determine the role of pyrimidine cyclobutane dimers in UV light-induced mutagenesis in monkey cells. The vector DNA was UV irradiated and then introduced into monkey cells by transfection. After replication, vector DNA was recovered from the cells and tested for mutations in its supF suppressor tRNA marker gene by transformation of Escherichia coli carrying a nonsense mutation in the beta-galactosidase gene. When the irradiated vector was treated with E. coli photolyase prior to transfection, pyrimidine cyclobutane dimers were removed selectively. Removal of approximately 90% of the pyrimidine cyclobutane dimers increased the biological activity of the vector by 75% and reduced its mutation frequency by 80%. Sequence analysis of 72 mutants recovered indicated that there were significantly fewer tandem double-base changes and G X C----A X T transitions (particularly at CC sites) after photoreactivation of the DNA. UV-induced photoproducts remained (although at greatly reduced levels) at all pyr-pyr sites after photoreactivation, but there was a relative increase in photoproducts at CC and TC sites and a relative decrease at TT and CT sites, presumably due to a persistence of (6-4) photoproducts at some CC and TC sites. These observations are consistent with the fact that mutations were found after photoreactivation at many sites at which only cyclobutane dimers would be expected to occur. From these results we conclude that UV-induced pyrimidine cyclobutane dimers are mutagenic in DNA replicated in monkey cells.

DNA strand specificity for UV-induced mutations in mammalian cells.

“…Mutations present in only one of the investigated M13 clones of a mutant were probably caused by the Taq polymerase. We observed in 15,000 sequenced bases 16 mutations introduced by Taq polymerase, giving an overall error frequency of about 1 in 940 bases after 32 cycles of amplification.…”

“…This predominance has been explained to occur through preferential insertion of adenine opposite noncoding lesions by DNA polymerase(s) during DNA replication (21). Protic-Sabljic et al (16) showed that, when the ratio between pyrimidine dimers and photoproducts in the supF shuttle vector was decreased by specific removal of pyrimidine dimers from the plasmid DNA with photolyase in vitro before transfection to monkey cells, an increase in the proportion of transversions among the class of UV-induced base pair substitutions occurred. The differences in UV mutation spectra between that found at the hprt gene in repair-proficient V79 cells and that at the aprt gene in CHO cells could be due to a difference in rate and efficiency with which photoproducts are removed from these genes.…”

“…The two types of DNA damage which are considered to be primarily responsible for the lethal and mutagenic effects of UV irradiation are the cyclobutane pyrimidine dimer (7,19) and the (6-4) pyrimidine-pyrimidone photoproduct (3, 10). Still, controversy exists over the relative importance of both types of lesions for cell killing and mutagenesis (4,6,13,16).It has been shown for cultured Chinese hamster and human cells that removal of dimers from the transcriptionally active dhfr gene is more rapid than for the genome overall (1, 2, 11) and that possibly even a strand specificity in DNA repair exists (12). Dimers were removed at a faster rate in human cells and to a larger extent in Chinese hamster ovary (CHO) cells from the transcribed strand from the amplified dhfr gene than from the nontranscribed strand.…”

“…The two types of DNA damage which are considered to be primarily responsible for the lethal and mutagenic effects of UV irradiation are the cyclobutane pyrimidine dimer (7,19) and the (6-4) pyrimidine-pyrimidone photoproduct (3, 10). Still, controversy exists over the relative importance of both types of lesions for cell killing and mutagenesis (4,6,13,16).…”

Third strand-mediated psoralen-induced correction of the sickle cell mutation on a plasmid transfected into COS-7 cells

“…This difference is consistent with the known mutagenicity of UV irradiation of DNA, which results in error-prone repair of T-T dimers of various types. 34 As an additional control, plasmids with covalently attached delivery strand were transformed directly in E. coli in order to establish that the E. coli amplification step does not contribute to the overall mutagenesis or mutation correction. In fact, no mutation correction or any other sequence changes were detected.…”