Kinetics of Oxidized Cytosine Repair by Endonuclease III of Escherichia coli

Abstract

Endonuclease III of Escherichia coli excises a broad range of oxidized, hydrated and ring-fragmented pyrimidines from DNA. The kinetic parameters were compared for repair of three potentially mutagenic oxidized cytosine lesions: 5,6-dihydroxy-5,6-dihydro-2‘-deoxyuridine (uracil glycol or Ug), 5-hydroxy-2‘-deoxycytidine (5-ohC), and 5-hydroxy-2‘-deoxyuridine (5-ohU). Site-specifically modified 40-mer oligonucleotides containing each of the three lesions in the same sequence context were synthesized chemically or by a combination of chemical and enzymatic methods. Appropriately protected phosphoramidites of 5-ohC and 5-ohU were synthesized and incorporated into oligonucleotides by standard solid-phase synthetic methods. The lability of Ug made it necessary to use an alternative approach to prepare the analogous 40-mers containing Ug. An uracil containing pentamer oligonucleotide was oxidized with OsO₄ to generate the corresponding Ug containing product, which was then ligated into an oligonucleotide scaffold to generate 40 base pair duplexes. Using ³²P-labeled substrates and a gel electrophoresis based assay, the values of K_m and V_max for excision of 5-ohC, 5-ohU, and Ug were determined. In this experimental system, the order of repair efficiency is Ug > 5-ohC > 5-ohU based on ratios of V_max/K_m. Modest effects were observed when the base paired opposite the lesion was changed from G to A.