Genetic effects of thymine glycol: site-specific mutagenesis and molecular modeling studies.

Abstract

The mutational specificity and genetic requirements for mutagenesis by 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), one ofthe principal DNA lesions induced by oxidation and ionizing radiation, has been investigated in Escherichia coli. Thymine glycol was positioned at a unique site in the single-stranded genome of a bacteriophage M13mpl9 derivative. Replication of the genome in E. coli yielded targeted mutations at a frequency of 0.3%; the mutations were exclusively T -* C. Mutagenesis was independent of SOS and nth (nth encodes endonuclease Ill, a thymine glycol repair enzyme). The adduct was not detectably mutagenic in duplex DNA. A chemical rationalization for the mutation observed for thymine glycol was developed by applying molecular modeling and molecular mechanical calculations to the same DNA sequence studied in vivo. Modeling suggested that the 5R,6S isomer of cis-thymine glycol, when not base paired, was displaced laterally by -0.5 A toward the major groove in comparison to the position that thymine would otherwise occupy. This perturbation of DNA structure should increase the likelihood of a guanine-thymine glycol wobble base pair during replication, which would explain the mutational specificity ofthe base observed in the genetic experiments.The hydroxyl radical is the principal reactive species responsible for DNA damage by ionizing radiation in aerated aqueous solution (1-3). This and other reactive oxygen species are also generated in vivo during normal metabolism (3, 4). Despite some differences, the DNA damages produced by ionizing radiation and cellular oxidation are qualitatively similar (5), with thymine bases being most susceptible to modification (6, 7). The major stable product of thymine modification in vitro and in vivo is 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol or t'; predominantly the cis isomers) (7,8). Several studies have shown that t' inhibits DNA synthesis in vitro in most sequences (9-12), although certain local contexts allow DNA polymerases to bypass the lesion (12).The mutational specificity of ionizing radiation has been studied in bacteria and in mammalian cells (13)(14)(15)(16)(17)(18)(19). Base substitutions are the most frequently detected mutational event and, although there is no specificity for any one substitution, most studies agree that the major mutational change is the G-C A-T transition. However, it is estimated that '40% of all ionizing radiation-induced mutations in Escherichia coli occur at AT sites.$ Moreover, Salmonella typhimurium strains with deletions in oxyR, a gene that positively controls a regulon affording protection against oxidative damage, exhibit a marked enhancement of spontaneous mutations such as T-A --AT transversions (20). Together, these data show that a significant fraction of oxidant-and radiation-induced mutations occurs at A-T base pairs, and it was the goal of our work to determine the contribution of t' to this component of the mutational spectrum.MATERIALS AND METHODS Materials. E. coli strain MM294A (lac') was...

Stimulated human leukocytes cause activating mutations in the K-ras proto-oncogene

“…Since the ®delity of replication of . OH-modi®ed DNA bases can vary greatly depending on the type of DNA polymerase employed, the surrounding primary DNA sequences, and/or whether the DNA template is single-or doublestranded (Kuchino et al, 1987;Shibutani et al, 1991;Basu et al, 1989;Kunkel and Alexander, 1986); the di erences between our results and these previous studies are not surprising. Indeed, these di erences underscore the importance of utilizing mammalian cells, double-stranded DNA, and genes that are relevant to human carcinogenesis as model systems for these types of studies.…”

“…OH-modi®ed DNA bases, including 8 hydroxydeoxyguanosine (Kuchino et al, 1987;Shibutani et al, 1991;Wood et al, 1990;Moriya et al, 1991;Cheng et al, 1992;Moriya, 1993), cytosine glycol (Hayes et al, 1988), thymine glycol (Basu et al, 1989), and 8 hydroxydeoxyadenosine (Guschlbauer et al, 1991), to mispair with each of the four DNA bases. The spectrum of mutations observed in our .…”

Structure of a Duplex DNA Containing a Thymine Glycol Residue in Solution

“…The presence of thymine glycol is a block to replication (23)(24)(25)(26)(27)(28). Studies of the effects of thymine glycol on the replication of M13 in vitro using polymerase I, E. coli DNA polymerase I, and T4 polymerases indicate that thymine glycol stops replication either one residue before or at the site of damage.…”

“…Thus, there may be as many as three routes to thymine glycol repair: endo III; exonuclease III and endo IV; and SOS (13). These results indicate that thymine glycol sites can be repaired in vivo in organisms ranging from E. coli to mammals.The presence of thymine glycol in DNA can have profound consequences on DNA replication (9,13,(23)(24)(25)(26)(27). The presence of thymine glycol is a block to replication (23-28).…”

“…The presence of thymine glycol in DNA can have profound consequences on DNA replication (9,13,(23)(24)(25)(26)(27). The presence of thymine glycol is a block to replication (23)(24)(25)(26)(27)(28).…”