Kinetics of Oxidized Cytosine Repair by Endonuclease III of Escherichia coli

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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 Km and Vmax 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 Vmax/Km. Modest effects were observed when the base paired opposite the lesion was changed from G to A.

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