Excision of 5,6-dihydroxy-5,6-dihydrothymine, 5,6-dihydrothymine, and 5-hydroxycytosine from defined sequence oligonucleotides by Escherichia coli endonuclease III and Fpg proteins: Kinetic and mechanistic aspects

Oligonucleotides that contain a single modified pyrimidine, i.e., thymine g lycol (Tg), 5,6-dihydrothymine (DHT), and 5-hydroxycytosine (5-OHC) were sy nthesized in order to investigate the substrate specificity and the excisio n mechanism of two Escherichia coli repair enzymes: endonuclease III and fo rmamidopyrimidine DNA glycosylase (Fpg). Three techniques of analysis were employed. A gas chromatography-mass spectrometry (GC-MS) assay with HPLC pr epurification was used to quantify the release of the modified bases, while polyacrylamide gel electrophoresis and matrix-assisted laser-desorption io nization-mass spectrometry (MALDI-MS) provided insights into the mechanism of oligonucleotide cleavage. Values of V-m/K-m constants lead to the conclu sion that the substrates are processed by endonuclease III with the followi ng preference: Tg much greater than 5-OHC > DHT. This confirms that Tg is a n excellent substrate for endonuclease III. Fpg-mediated cleavage of the 5- OHC-containing oligonucleotide is processed at the same rate than endonucle ase III. Furthermore, Fpg was found to have a little but relevant activity on DHT-containing oligonucleotide, thus broadening the substrate specificit y of this enzyme to a new modified pyrimidine. While 5-OHC-containing oligo nucleotides are cleaved by the two enzymes, no or a small amount of the mod ified base was found to be released, as determined by GC-MS. From these dat a it may be suggested that 5-OHC could be modified during its enzymatic exc ision. Finally, MALDI-MS analyses shed new light on the mechanism of action of endonuclease III: the molecular masses of the repaired fragments of 5-O HC- and DHT-containing oligonucleotides showed that endonuclease III cleave s the DNA backbone mainly through a hydrolytic process and that no beta-eli mination product was detected.