Repair and coding properties of 5-hydroxy-5-methylhydantoin nucleosides inserted into DNA oligomers

1-(2-Deoxy-beta-D-erythro-pentofuranosyl)-5-hydroxy-5-methylhydantoin (5-OH -5-Me-dHyd) (3) has been shown to be a major oxidation product of thymidine formed upon exposure of DNA to (OH)-O-.-radical and excited photosensitize rs. To investigate the biological and structural significance of the 6-OH-5 -Me-dHyd residue to DNA, the latter modified 2'-deoxyribonucleoside was che mically prepared and then site-specifically incorporated into oligo deoxyri bonucleotides. This was efficiently achieved using the phosphoramidite appr oach that involved mild deprotection conditions. The purity and the integri ty of the modified synthetic DNA fragments were checked using different com plementary techniques such as HPLC and polyacrylamide gel electrophoresis, together with electrospray ionization and MALDI-TOF mass spectrometry. The piperidine test applied to 5-OH-5-Me-dHyd containing oligonucleotides showe d a weak instability of hydantoin nucleoside inserted into the oligonucleot ide chain. Several enzymatic experiments aimed at determining the biochemic al features of such a DNA lesion were carried out. Thus, processing of 5-OH -5-Me-dHyd by nuclease P-1, snake venom phosphodiesterase, and calf spleen phosphodiesterase was investigated. The specificity and the mechanism of ex cision of the lesion by several bacterial and yeast DNA N-glycosylases, nam ely, endonuclease III (endo III), endonuclease VIII (endo VIII), formamidop yrimidine DNA N-glycosylase (Fpg), Ntg1 protein (Ntg1), Ntg2 protein (Ntg2) , and Ogg1 protein (yOgg1), were also determined. These repair studies clea rly showed that all these enzymes, with the exception of the yOgg1 protein, are able to recognize and remove 5-hydroxy-5-methylhydantoin from the doub le-stranded DNA fragment. Finally, a 22-mer DNA oligomer bearing a 5-OH-5-M e-dHyd residue was used as a template to study the in vitro nucleotide inco rporation opposite the damage by the Klenow fragment of Escherichia coli po lymerase I, Taq DNA polymerase, and DNA polymerase beta. Thus, it may be co ncluded that the oxidized thymine residue is a strongly blocking lesion for the three studied DNA polymerases.