Replication through an abasic DNA lesion: structural basis for adenine selectivity

Abstract

Abasic sites represent the most frequent DNA lesions in the genome that have high mutagenic potential and lead to mutations commonly found in human cancers. Although these lesions are devoid of the genetic information, adenine is most efficiently inserted when abasic sites are bypassed by DNA polymerases, a phenomenon termed A-rule. In this study, we present X-ray structures of a DNA polymerase caught while incorporating a nucleotide opposite an abasic site. We found that a functionally important tyrosine side chain directs for nucleotide incorporation rather than DNA. It fills the vacant space of the absent template nucleobase and thereby mimics a pyrimidine nucleobase directing for preferential purine incorporation opposite abasic residues because of enhanced geometric fit to the active site. This amino acid templating mechanism was corroborated by switching to pyrimidine specificity because of mutation of the templating tyrosine into tryptophan. The tyrosine is located in motif B and highly conserved throughout evolution from bacteria to humans indicating a general amino acid templating mechanism for bypass of non-instructive lesions by DNA polymerases at least from this sequence family.

Figure 1

Nucleotide incorporation opposite an abasic site. (A) Hydrolysis of the glycosidic bond leading to nucleobase loss and formation of abasic site AP. B: nucleobase. (B) Structure of the abasic site analogue F. (C) Partial primer template sequence used in primer extension experiments. (D) Single nucleotide incorporation of KlenTaq opposite F for 2, 10, or 60 min, respectively. The respective dNTP is indicated.

Figure 2

Structures of KlenTaq in complex with substrates. (A) Structure of KlenTaq_AP (purple) superimposed with KlenTaq (green, PDB 1QSY) bound to undamaged DNA. The location of the O helix is indicated. (B) Close-up view highlights the location of the O and N helices in KlenTaq_AP. (C) Superimposition of KlenTaq_AP, KlenTaq (green, PDB 1QSY, closed conformation) and KlenTaq (orange, PDB 2KTQ, open conformation).

Figure 3

Interaction network of incoming ddATP opposite abasic site F. (A) Hydrogen bond network stabilizing ddATP. Labelled are the amino acid side chains R587 and Y671. (B) Superimposed structures of KlenTaq_AP (purple) and KlenTaq (green, PDB 1QSY) show the difference in orientation of residues R587 and Y671. (C) Inner coordination spheres showing metal ion in KlenTaq_AP. A Mg²⁺ ion (purple sphere) is coordinated by the triphosphate moiety of ddATP and two water molecules (red spheres). Two additional water molecules form hydrogen bonds to residues D610, Y611, and D785. Residue K663, recently, discussed to act as general acid in catalysis, is shown. (D) KlenTaq (PDB 1QSY) showing the same residues as in (C). All distances are in Å.

Figure 4

Active site and nascent base pair assemblies. (A) Close-up view of KlenTaq_AP active site processing ddATP opposite abasic site F. Shown are O helix, residues Y671, and F667, the respective template residues and incoming ddATP. (B) Structure of KlenTaq (PDB 1QSY) active site processing ddATP opposite template dT. (C) Top view to the nascent base pair opposite F. In the front the incoming ddATP opposite Y671 is depicted. The hydrogen bond between the hydroxyl group of Y671 and N3 of adenine is indicated as dashed line. In transparent the first nucleobase pair of the primer template terminus is shown. (D) Top view of the nascent base pair in KlenTaq (PDB 1QSY). The hydrogen bonding between the incoming ddATP and the templating dT is shown in dashed lines. The primer template terminus is illustrated in transparent.

Figure 5

Nucleotide incorporation opposite abasic site F. (A) Single nucleotide incorporation of KlenTaq Tyr671Trp mutant opposite F for 2, 10, or 60 min, respectively. The respective dNTP is indicated. (B, C) Pre-steady-state kinetics of single nucleotide incorporation opposite F catalysed by KlenTaq wild type (B) and Tyr671Trp mutant (C), respectively. The curves show dependence of the observed pre-steady-state rates (k_obs) on dNTP concentration. The k_obs values were plotted versus the concentration of the used dNTP and fitted to a hyperbolic equation. (D) Amino acid sequence alignment of DNA polymerases highlighting the conserved position equivalent to Y671 in KlenTaq.

Figure 6

Nucleotide incorporation at a blunt-end primer template duplex. (A) Partial sequence used in primer extension experiments. (B) Single nucleotide incorporation of KlenTaq by extension of blunt-end primer template duplex for 2, 10, or 60 min, respectively. The respective dNTP is indicated. (C) As in (B) for KlenTaq mutant Tyr671Trp in presence of a blunt-end primer template duplex.

Figure 7

Comparison of KlenTaq_BE (cyan) and KlenTaq_AP (purple). (A) Superimposed structures of KlenTaq_BE and KlenTaq_AP. (B) Comparison of the inner coordination sphere of metal ion in KlenTaq_BE and KlenTaq_AP. (C) Superimposition of the top view of the nascent nucleobase pair of KlenTaq_BE and KlenTaq_AP. (D) Close-up views of the active sites of KlenTaq_BE and KlenTaq_AP. Labelled are O helix, residues F667, and Y671.