Replication in vitro and cleavage by restriction endonuclease of 5-formyluracil- and 5-hydroxymethyluracil-containing oligonucleotides

Purpose: To investigate the biological consequences of 5-formyluracil (5-fo U) and 5-hydroxymethyluracil (5-hmU). Materials and methods: The authors constructed 22-mer origonucleotides cont aining a 5-foU or 5-hmU residue at the same sites. The effects of such modi fications on the ability to serve as a template for DNA polymerase and on t he cleavage by sequence-specific restriction endonuclease were examined. Results: The Klenow fragment of DNA polymerase I and Thermus thermophilus D NA polymerase read through the sites of 5-foU and 5-hmU in the templates. 5 -FoU directed the incorporation of dCMP in addition to dAMP opposite the le sion during DNA synthesis. The DNA polymerases incorporated only dAMP oppos ite the 5-hmU. The substitution of thymine by 5-foU within the recognition site of the restriction endonucleases HincII and Sail inhibited or prevente d the cleavage by the enzymes, whereas the enzymes cleaved the 5-hmU-contai ning oligonucleotides at the same rate as the T-containing oligonucleotides . Conclusions: These results indicated that the 5-foU-A base pair is less sta ble than the T-A base pair and that 5-foU can form a base pair with C in ad dition to A. It was also demonstrated that the oxidation of thymine to 5-hm U does not result in substantial deterioration.