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-A 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.

Fig. 1.

Tg is the product of ionizing radiation and oxidation. γ-irradiation produces equal amounts of the (5R, 6S) and (5S, 6R) cis isomers, whereas oxidation generates preferentially the (5R, 6S) isomer.

Fig. 2.

RB69 gp43 stalls after incorporation across Tg site. (a) Wild-type RB69 gp43 is blocked after incorporation across Tg, even after an incubation of 5 min. (b) With an exonuclease-deficient DNA polymerase (D222A/D327A), Tg is a strong pause site.

Fig. 3.

Tg base-pairs with A and maintains minor groove interactions. (a) Tg·acyAMP base pair with overlaid 2.65-Å simulated annealing omit map contoured at 5 σ. Hydrogen bonds are indicated by black dotted lines. (b) The Tg·acyAMP structure (green) was superimposed onto an RB69 gp43 complex obtained with undamaged DNA (29) (yellow; PDB ID code 1IG9). The Tg·acyAMP base pair (green) maintains the same water-mediated minor groove interaction with Y567 as the normal G·C base pair (yellow), and the universal hydrogen-bond acceptors O2 and N3 (33) occupy the same position in the two superimposed base pairs. The water molecules are shown as red spheres.

Fig. 4.

Translocation occurred after incorporation of A opposite Tg. The letter “i” indicates the position of the insertion site, and “n-1” and “n-2” are the postinsertion sites after one or two cycles of incorporation and translocation. (a) The Tg·acyAMP base pair is located in the postinsertion site (n-1). (b) The F·dAMP base pair, which did not translocate, is in the insertion site (i). (c) The complex with undamaged DNA is shown for comparison. The A·dTTP base pair is in the insertion site.

Fig. 5.

Interaction of Tg with surrounding bases. Superposition of the Tg·acyAMP complex (green) with an incipient base pair (A·dTTP) from an RB69 gp43 complex with normal DNA (29) (yellow; PDB ID code 1IG9). The adenine (yellow) overlaid on the Tg·acyAMP structure (green) illustrates that the methyl group of Tg would sterically clash with the six-member ring of any purine base in the insertion site (i). The displaced 5′-guanine (green) rotates out of the way and is stabilized by two hydrogen bonds with Tg. Tg also interacts with the 3′-guanine via a water-mediated interaction. Water molecules are shown as red spheres.