PNA-Based Reagents for the Direct and Site-Specific Synthesis of Thymine Dimer Lesions in Genomic DNA

摘要

UV irradiation of cells furnishes large numbers of mutagenic cyclobutane pyrimidine dimer lesions (CPD,dT=dT) as a consequence of [2pi + 2PI] cycloaddition reactions between adjacent thymidines.In recent years,the need for DNA containing such lesions at defined sites has steadily grown in order to allow biochemical,crystallographic,and cellular studies.These are needed to learn how DNA lesions induce mutations and how cells remove lesions from the genome.Particularly,DNA with dT=dT lesions is needed because they are repaired by various pathways,shifting them into the center of DNA repair.The chemical synthesis of DNA containing dT=dT dimers is a time-consuming process.It requires synthesis of a dimer lesion phosphoramidite building block,chemical synthesis of DNA using this building block,purification of the prepared DNA,and,if required,ligation of the DNA into a larger DNA fragment.Alternatively,small DNA fragments with one dTpdT dinucleotide site can be irradiated in the presence of acetophenone to give dT=dT containing DNA and with side products that need to be separated by HPLC.Both methods are labor intensive,and it is difficult to create DNA strands with more than one thymine dimer.