Processing of Thymine Glycols, Urea Residues and AP Sites in Escherichia Coli

Abstract

In order to examine the biological consequences of oxidative DNA damage, we have selectively introduced damages of interest into phage transfecting DNA so that we can examine the repair of these lesions in vivo. Thymine glycol is a relatively stable ring saturation product of thymine that is formed as a consequence of hydroxyl radical attack on the 5,6 double bond. Thymine glycol is found in DNA X-irradiated in vitro (1,2) and in vivo (3,4) and appears to be formed as a consequence of oxidative stress (5). Thymine glycol is a replicative block to DNA polymerases in vitro (6–9), however, it retains pairing capacity since an A is inserted opposite the lesion before synthesis is arrested (10). Urea residues are fragmentation products of thymine hydroperoxides and are found as stable residues attached to the backbone of irradiated DNA (1). Urea residues are also in vitro replication blocks to DNA synthesis, however, since urea residues are totally noninstructive, synthesis is arrested one base before the damage (6,8). Apurinic/apyrimidinic (AP) sites are common DNA lesions produced by radiation and chemicals and are also formed as intermediates in repair processes initiated by DNA glycosylases (11). AP sites are noninstructive lesions that are also blocks to DNA replication in vitro (12). Thymine glycols, urea residues and AP sites are easily quantitated in DNA by enzymatic and immunochemical procedures (13,14). Most importantly, thymine glycols can be selectively introduced into DNA by osmium tetroxide oxidation (15,16); urea residues can be formed by alkali hydrolysis of thymine glycol-containing DNA (17); and AP sites can be introduced by heat/acid treatment (18).