Mutagenicity of a unique thymine-thymine dimer or thymine-thymine pyrimidine pyrimidone (6-4) photoproduct in mammalian cells

Abstract

The mutagenic properties of UV-induced photoproducts, both the cis-syn thymine-thymine dimer (TT) and the thymine-thymine pyrimidine pyrimidone (6-4) photoproduct [T(6-4)T] were studied in mammalian cells using shuttle vectors. A shuttle vector able to replicate in both mammalian cells and bacteria was produced in its single-stranded DNA form. A unique photoproduct was inserted at a single restriction site and after recircularization of the single-stranded DNA vector, this latter was transfected into simian COS7 cells. After DNA replication the vector was extracted from cells and used to transform bacteria. Amplified DNA was finally analyzed without any selective screening, DNA from randomly picked bacterial colonies being directly sequenced. Our results show clearly that both lesions are mutagenic, but at different levels. Mutation frequencies of 2 and 60% respectively were observed with the TT dimer and the T(6-4)T. With the TT dimer the mutations were targeted on the 3'-T. With the T(6-4)T a large variety of mutations were observed. A majority of G-->T transversions were semi-targeted to the base before the 5'-T of the photoproduct. These kinds of mutations were not observed when the same plasmid was transfected directly into SOS-induced JM105 bacteria or when the T(6-4)T oligonucleotide inserted in a different plasmid was replicated in SOS-induced SMH10 Escherichia coil bacteria. These semi-targeted mutations are therefore the specific result of bypass of the T(6-4)T lesion in COS7 cells by one of the eukaryotic DNA polymerases.

Genomic assay reveals tolerance of DNA damage by both translesion DNA synthesis and homology-dependent repair in mammalian cells

“…This is consistent with the results that we have previously obtained with nonreplicating gapped plasmids in mammalian cells (28,41) and with an earlier report using a replicative plasmid (42). A recent study reported that TLS across a TT 6-4 PP in a replicating plasmid is largely accurate (43).…”

“…A recent study reported that TLS across a TT 6-4 PP in a replicating plasmid is largely accurate (43). This difference does not stem from a replicative vs. a gapped nonreplicating plasmid as those authors suggested, because an earlier study using a replicative plasmid found that TLS is highly mutagenic (42), like in our study. The reason for the difference is not known yet, but it should be pointed out that the TLS that we have monitored across chromosomal TT 6-4 PP was highly mutagenic.…”

Enhanced DDB2 Expression Protects Mice from Carcinogenic Effects of Chronic UV-B Irradiation

“…Most notably, an elevated UV-induced skin cancer proneness has been found for XPCÀ/À mice (29), which are TCR proficient and differ from the WT mice by the absence of 6-4PP GGR (20). In this respect, it is worth noting that mutagenic bypass of 6-4PPs in mammalian cells has been reported to be >10 times higher than for CPDs (46). The similar delay in repair of 6-4PPs in both human and mouse cells deficient for DDB2 suggests that the accelerating role of DDB2 in 6-4PP repair might also be important in the protection of humans against UV-induced skin cancer.…”

Recognition and Repair of Compound DNA Lesions (Base Damage and Mismatch) by Human Mismatch Repair and Excision Repair Systems

“…2). However, most of the UV-induced mutations at TT sequences are T-A-to-C-G transitions at the 3Ј T-A base pair (19,32). Therefore, in addition to the 5Ј T-G mismatches in our TϽϾT compound lesions, we wished to use 3Ј T-G-associated compound lesions to ascertain that the preferential repair of compound TϽϾT lesions contributes to UV mutagenesis.…”