Analysis of transcription-coupled repair (TCR) of oxidative lesions here reveals strand-specific removal of 8-oxo-guanine (8-oxoG) and thymine glycol both in normal human cells and xeroderma pigmentosum (XP) cells defective in nucleotide excision repair. In contrast, Cockayne syndrome (CS) cells including CS-B, XP-B/CS, XP-D/CS, and XP-G/CS not only lack TCR but cannot remove 8-oxoG in a transcribed sequence, despite its proficient repair when not transcribed. The XP-G/CS defect uniquely slows lesion removal in nontranscribed sequences. Defective TCR leads to a mutation frequency at 8-oxoG of 30%-40% compared to the normal 1%-4%. Surprisingly, unrepaired 8-oxoG blocks transcription by RNA polymerase II. These data imply that TCR is required for polymerase release to allow repair and that CS results from defects in TCR of oxidative lesions.
The processing of a unique 8-oxoguanine residue in DNA has been studied in mammalian cells using a single-stranded shuttle vector. A fragment of human Ha-ras carrying the lesion on the first (G1) or the second guanine (G2) of codon 12 was inserted in a shuttle plasmid. Extrachromosomal DNA is replicated in animal cells, extracted and used to transform bacteria to be amplified and individualized. DNA sequencing of bacterial clones showed the mutagenic potency of 8-oxoguanine in vivo to be approximately 4%. The presence of the 8-oxoguanine does not greatly affect survival of the progeny. No significant difference was observed between the mutation frequencies induced by 8-oxoguanine located either at the G1 or G2 position. The majority of the mutations, targeted at the lesion level, are G to T transversions. These base substitutions induced respectively glycine to cysteine (G1) or valine (G2) change in the P21ras protein. These mutations may contribute to activation of the protooncogene, leading to spontaneous tumorigenesis.
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.
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