High resolution mapping of UV-induced photoproducts in the Escherichia coli lacI gene. Inefficient repair of the non-transcribed strand correlates with high mutation frequency.

摘要: UV-induced DNA photoproduct formation and repair has been examined at the gene nucleotide level in Escherichia coli using two newly developed quantitative assays. A multiplex PCR assay was used to measure both constitutive lacI inducible lacZ gene, simultaneously. Both genes displayed similar frequencies (0.4 lesions/kb per 100 J/m2). Following a 15 minute recovery period, 36% 39% of damage resulting from J/m2 removed genes, respectively. Under growth conditions applied, expressed very low rate 0.3% beta-galactosidase activity as compared induced cells. reiterative primer extension employed examine level. Analysis photoproducts first 184 base-pairs genomic E. revealed that are linearly with dose slope is sequence context-dependent. post-irradiation period differences efficiency individual nucleotides. Repair on transcribed strand generally twice efficient non-transcribed strand, indicating strand-specific occurs constitutively coli. Comparison distribution an established mutation spectrum wild-type cells form all mutagenic hotspots. Some sites frequency mutations were not observed be formation. However, appeared mutagenic. This especially true for those efficiently repaired strand. It hypothesized preferential this may prevent many these becoming These data strongly support hypothesis arise inefficiently nontranscribed