Oxidized abasic residues in DNA constitute a major class of radiation and oxidative damage. Free radical attack on the nucleotidyl C-1 carbon yields 2-deoxyribonolactone (dL) as a significant lesion. Although dL residues are efficiently incised by the main human abasic endonuclease enzyme Ape1, we show here that subsequent excision by human DNA polymerase ␤ is impaired at dL compared with unmodified abasic sites. This inhibition is accompanied by accumulation of a protein-DNA cross-link not observed in reactions of polymerase ␤ with unmodified abasic sites, although a similar form can be trapped by reduction with sodium borohydride. The formation of the stably cross-linked species with dL depends on the polymerase lysine 72 residue, which forms a Schiff base with the C-1 aldehyde during excision of an unmodified abasic site. In the case of a dL residue, attack on the lactone C-1 by lysine 72 proceeds more slowly and evidently produces an amide linkage, which resists further processing. Consequently dL residues may not be readily repaired by "shortpatch" base excision repair but instead function as suicide substrates in the formation of protein-DNA crosslinks that may require alternative modes of repair.Mutagenesis and disruption of the cell cycle caused by DNA damage is counteracted by DNA repair systems. In the base excision repair pathway (1-3), DNA glycosylases eliminate damaged bases to generate abasic (AP) 1 sites, which are also formed in large numbers by spontaneous depurination (2). In either case, AP sites are incised by an AP endonuclease to allow subsequent DNA repair synthesis and excision of the abasic residue. In mammalian cells, incision is carried out by the major AP endonuclease Ape1 protein (also called Apex, Hap1, or Ref1), while the excision step for regular abasic residues is thought to be mainly carried out by DNA polymerase ␤ (Pol␤) using a ␤-elimination mechanism. A distinct branch of the base excision pathway involves strand displacement repair synthesis and excision of the displaced, damaged strand by the FEN1 nuclease (4 -6). Still another variation is potentiated by the initial DNA glycosylase (7) because some of these enzymes carry out a second reaction to cleave at the abasic site by ␤- elimination (1, 3). The resulting 3Ј-blocked products must then be removed by an enzyme such as Ape1 before repair synthesis can proceed (1).Base excision repair acts on a wide variety of deaminated, alkylated, or oxidized bases (2, 3). However, oxidative damage to DNA also produces various modified abasic residues that may complicate the repair scenario (1). For example, free radical attack forms strand breaks with fragmentary or oxidized products of deoxyribose; when these are present at the 3Ј terminus, removal by Ape1 may be the rate-limiting repair step (8, 9). Oxidized abasic residues without direct strand breakage (10) include 2-deoxypentos-4-ulose residues (a major lesion produced by the antitumor drug bleomycin) and 2-deoxyribonolactone (dL) residues (formed by diverse oxidative agents). 2-D...

Copper–phenanthroline complexes oxidatively damage and cleave nucleic acids. Copper bis-phenanthroline and copper complexes of mono- and bis-phenanthroline conjugates are used as research tools for studying nucleic acid structure and binding interactions. The mechanism of DNA oxidation and cleavage by these complexes was examined using two copper–phenanthroline conjugates of the sequence-specific binding molecule, distamycin. The complexes contained either one or two phenanthroline units that were bonded to the DNA-binding domain through a linker via the 3-position of the copper ligand. A duplex containing independently generated 2-deoxyribonolactone facilitated kinetic analysis of DNA cleavage. Oxidation rate constants were highly dependent upon the ligand environment but rate constants describing elimination of the alkali-labile 2-deoxyribonolactone intermediate were not. Rate constants describing DNA cleavage induced by each molecule were 11–54 times larger than the respective oxidation rate constants. The experiments indicate that DNA cleavage resulting from β-elimination of 2-deoxyribonolactone by copper–phenanthroline complexes is a general mechanism utilized by this family of molecules. In addition, the experiments confirm that DNA damage mediated by mono- and bis-phenanthroline copper complexes proceeds through distinct species, albeit with similar outcomes.

Copper-phenanthroline complexes and their conjugates are useful reagents for studying nucleic acid interactions. Although DNA cleavage by such complexes was discovered more than 20 years ago, significant questions remain unanswered regarding the chemical mechanism(s) by which DNA is damaged. Kinetic evidence is provided, which demonstrates that the major pathway for DNA damage by a minor groove binding molecule conjugated to copper phenanthroline (6) involves C1'-oxidation. Additional experiments using 6 and a DNA substrate containing 2-deoxyribonolactone (1) show that direct strand breaks are produced via beta-elimination from 1. These studies support the original mechanism for DNA damage by copper phenanthroline put forth by Sigman and a more recent proposal concerning the mechanism for direct strand break formation.