Establishment of a Method for Analyzing Translesion DNA Synthesis across a Single Bulky Adduct in Human Cells

摘要

Translesion DNA synthesis (TLS) is a tolerance pathway of replication block caused by DNA lesions. To measure the efficiency and fidelity of TLS in human cells, we established a shuttle vector assay by modifying of a bacterial TLS assay system. The assay consists of transfection of DNA repair-deficient human cells with a plasmid possessing a single DNA adduct, and transformation of indicator bacteria with plasmids extracted from the cells. We show that plasmid replication was suppressed to 1/9 by a single aminobiphenyl-dG adduct, and the mutant frequency of TLS-operated plasmids was 0.31, of which the major mutation (78%) was G to T transversion. The results demonstrate that this assay is applicable in practice for investigating TLS in human cells.