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.

Amino Acid Templating Mechanisms in Selection of Nucleotides Opposite Abasic Sites by a Family A DNA Polymerase

“…S1) have shown that purines, in particular adenosine, and to a lesser extent guanosine, are most frequently incorporated opposite the lesion. The strong preference for adenosine has been termed "A-rule" (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).…”

“…Superior stacking as well as solvation properties of adenine have been discussed as the driving force behind adenine selection (17, 29 -31). However, previously reported structures of KlenTaq, the large fragment of Thermus aquaticus (Taq) DNA polymerase I, which is a member of the sequence family A, suggests that this enzyme follows the A-rule by applying an amino acid templating mechanism (19,20). Thereby, interaction with Tyr-671 seems to be the basis of the preference of purines.…”

“…It facilitates nucleotide incorporation by mimicking a pyrimidine nucleobase directing for purine incorporation opposite abasic sites due to the enhanced geometric fit to the active site (32,33). The crucial role of this tyrosine in translesion synthesis, which is highly conserved throughout evolution in DNA polymerase family A from bacteria to humans (34), was further probed by analysis of site-directed mutations (19). Interestingly, when the six-membered ring of tyrosine was mutated to the bicyclic indole of tryptophan, the A-rule was shifted to a "C/T-rule," further supporting geometric factors as determinants of nucleotide selection opposite an abasic site.…”

Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase

“…The large fragment of Thermus aquaticus (Taq) DNA polymerase (in short KlenTaq, N-terminally truncated form of Taq polymerase) was chosen as the target because this enzyme class is heavily employed and well characterized on a functional and structural level (24)(25)(26)(27)(28)(29)(30)(31). We compared two dNTP analogsnamely dT spin TP and dT dend TP (Fig.…”

Two-dimensional IR spectroscopy of the anti-HIV agent KP1212 reveals protonated and neutral tautomers that influence pH-dependent mutagenicity

“…As the mutagenicity of protonated KP1212 is insufficiently explained by its predominant tautomeric form, we propose three other models by which the polymerase replicates across the lesion. One possibility involves the "A rule," where a polymerase replicating across a bulky damaged base or an abasic site is more likely to place an adenine in the opposing strand (33). The charged state of KP1212 may promote its ability to flip out of the helix stack, presenting the polymerase an intermediate structure reminiscent of an abasic site (Fig.…”