A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol

Abstract

Thymine glycol (Tg) is a common product of oxidation and ionizing radiation, including that used for cancer treatment. Although Tg is a poor mutagenic lesion, it has been shown to present a strong block to both repair and replicative DNA polymerases. The 2.65-Å crystal structure of a binary complex of the replicative RB69 DNA polymerase with DNA shows that the templating Tg is intrahelical and forms a regular Watson-Crick base pair with the incorporated A. The C5 methyl group protrudes axially from the ring of the damaged pyrimidine and hinders stacking of the adjacent 5 template guanine. The position of the displaced 5 template guanine is such that the next incoming nucleotide cannot be incorporated into the growing primer strand, and it explains why primer extension past the lesion is prohibited even though DNA polymerases can readily incorporate an A across from the Tg lesion.oxidative DNA damage ͉ structure ͉ DNA replication T hymine glycol (5,6-dihydro-5,6-dihydroxythymine; Tg), the most common oxidation product of thymine, is produced endogenously as a consequence of aerobic metabolism or via exogenous factors such as chemical oxidants or ionizing radiation (Fig. 1). It is estimated that 400 Tgs are formed per cell per day (1, 2), and the presence of Tg in DNA has been used as a marker for oxidative stress (1, 3). Moreover, Tg is one of the predominant types of base modifications produced by ionizing radiation (4, 5), including that used in cancer therapy.Of the oxidatively modified DNA bases retaining an intact ring, Tg is thought to induce the most distortion in the regular structure of DNA. Even though there are DNA repair enzymes specialized in excising Tg from the genome, such as human , statistically a few of the damaged bases will evade repair, which means that DNA polymerases will encounter these lesions during replication. Although Tg is a poor mutagenic lesion because it generally pairs with A (9), it has been shown to be a very effective block to DNA replication (10-13). When Tg is encountered as a templating base by replicative or repair polymerases, termination of primer extension occurs immediately past the lesion site with A inserted opposite the Tg. Even in the presence of proofreading, extension proceeds no further. This finding contrasts with the situation observed when the lesion is an abasic site, where, in the presence of proofreading, termination sites are observed one base before the lesion (14). Therefore, it is extension past the Tg⅐adenine pair, rather than insertion across Tg, that constitutes the block to the replicative polymerases. Because Tg is a strong block to repair and replicative DNA polymerases in vitro (10-13), not surprisingly, it is a lethal lesion in vivo (9,(15)(16)(17)(18).Unlike normal DNA bases, Tg is nonplanar because of the loss of aromatic character that accompanies the addition of hydroxyl groups at the 5 and 6 positions of the ring (19). Computational simulations (20,21) predict that the axial orientation of the methyl group with respect to the pyrimidine r...

Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context

“…This is consistent with both biochemical and crystallographic studies from our laboratory with DNA polymerases that have shown that the methyl and hydroxyl groups present on the 5Ј position of thymine glycol severely alter stacking of the 5Ј base and in the case of guanine, a hydrogen bond is formed with Tg. Moreover, there is an additional interaction with a 3Ј-purine via a water molecule (64). Thus, in the case of the random sequence we used, 5Ј-G Tg G-3Ј, the Tg would be less able to be extruded into the Neil3 substrate binding pocket than in the telomere sequence context 5Ј-T Tg G-3Ј.…”

Structural Characterization of Viral Ortholog of Human DNA Glycosylase NEIL1 Bound to Thymine Glycol or 5-Hydroxyuracil-containing DNA

“…A local conformational perturbation may also be at play here. The presence of (5R,6S)-Tg within double-stranded DNA is known to hinder proper stacking of the 5Ј base, especially when that base is a purine (45,59,60). Furthermore, the nature of the base opposite the lesion was shown to modulate repair of Tg by human NEIL1 (49); Tg occupies the wobble position when mispaired with G (61) and is excised much more rapidly than Tg:A (49).…”

“…1) (24,26,43,44). Although both are oxidized pyrimidines, 5-OHU is planar, and Tg is not (45). Crystals of MvNei1E3Q with Tgcontaining DNA were obtained using crystallization conditions similar to those used for the complex of MvNei1 with DNA containing an abasic site analog (THF) (34).…”

Genetic Control of Replication through N1-methyladenine in Human Cells

“…It confers a 44º bend in the DNA helix and the 3ЈT is oriented perpendicular to the 5ЈT in the (6-4) TT photoproduct (14 -16). The Tg lesion also has no significant effect on the ability of oxidized T to form a correct base pair with an A; however, because of the addition of hydroxyl groups at C5 and C6 on Tg, the damaged base becomes non-planar and that prevents the base 5Ј to Tg from stacking above it (17)(18)(19)(20). Consequently, Tg presents a strong block to extension of synthesis from the Tg:A base pair.…”