Discrimination between translesion synthesis and template switching duringbypass replication of thymine dimers in duplex DNA

The goal of this study was to determine whether bypass replication occurs b y translesion synthesis or template switching (copy choice) when a duplex m olecule carrying a single cis,syn-cyclobutane thymine dimer is replicated i n vitro by human cell extracts. Circular heteroduplex DNA molecules were co nstructed to contain the SV40 origin of replication and a mismatch opposite to or nearby the dimer. Control molecules with only the mismatch were also prepared. Heteroduplexes were methylated at CpG islands and replicated in vitro (30 min). Following bisulfite treatment, the nascent DNA complementar y to the dimer-containing template was distinguished from the other three s trands by methylation-specific polymerase chain reaction. Cloning and seque ncing of polymerase chain reaction products revealed that 80-98% carried th e sequence predicted for translesion synthesis, with two adenines incorpora ted opposite the dimer. The fraction of clones with sequence predictive of template switching was reduced when extracts deficient in mismatch repair o r nucleotide excision repair activities were used to replicate the heterodu plex molecules. These results support the conclusion that lesion bypass dur ing in vitro replication of duplex DNA containing thymine dimers occurs by translesion synthesis.