Discrimination between translesion synthesis and template switching during bypass replication of thymine dimers in duplex DNA.
The goal of this study was to determine whether bypass replication occurs by translesion synthesis or template switching (copy choice) when a duplex molecule carrying a single cis,syn-cyclobutane thymine dimer is replicated in vitro by human cell extracts. Circular heteroduplex DNA molecules were constructed 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 complementary to the dimer-containing template was distinguished from the other three strands by methylation-specific polymerase chain reaction. Cloning and sequencing of polymerase chain reaction products revealed that 80-98% carried the sequence predicted for translesion synthesis, with two adenines incorporated opposite the dimer. The fraction of clones with sequence predictive of template switching was reduced when extracts deficient in mismatch repair or nucleotide excision repair activities were used to replicate the heteroduplex molecules. These results support the conclusion that lesion bypass during in vitro replication of duplex DNA containing thymine dimers occurs by translesion synthesis.
Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7525, USA.
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N
Nikolaishvili-Feinberg
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Cordeiro-Stone
D020137:Base Pair Mismatch; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018899:CpG Islands; D004247:DNA / Q000096:biosynthesis*; D019175:DNA Methylation; D017931:DNA Primers; D004260:DNA Repair; D004261:DNA Replication; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013447:Sulfites / Q000494:pharmacology; D013698:Templates, Genetic; D013941:Thymine / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D009692:Nucleic Acid Heteroduplexes; D013447:Sulfites; D004247:DNA; D013941:Thymine