Abstract
Adenine frequently pairs with the Hoogsteen edge of an oxidized guanine base (8OG) causing G to T transversions. The (syn)8OG:dA base pair is indistinguishable from the cognant base pair and can be extended by DNA polymerases with reduced efficiency. To examine the structural basis of this reduced efficiency, we sought to obtain the structure of the “product” complex of DNA polymerase (pol) β with the (syn)8OG:dA base pair at the primer terminus by soaking the binary complex crystals with a hydrolysable dCTP analogue complementary to the template base G. Crystallographic refinement of the structure revealed that the adenine of the (syn)8OG:dA base pair had been expelled from the primer terminus and a dCMP was inserted opposite 8OG in a reverse orientation; another uninserted molecule of the analogue was bound to the templating base G. This leads to an abortive complex that could form the basis of oxidatively-induced pol β stalling.
Highlights
Adenine frequently pairs with the Hoogsteen edge of an oxidized guanine base (8OG) causing G to T transversions
The 8OG:dA base pair is readily extended by most polymerases leading to G to T transversions, as the 8OG:dA base pair is not recognized as a lesion by the mismatch repair machinery or exonucleases; this is because of resemblance of the 8OG:dA base pair hydrogen bonding to that of a dT:dA base pair
Binary complex crystals with the 8OG:dA base pair at the primer terminus (n − 1 position) were grown; the templating base in the single-nucleotide gapped DNA was the natural dG base These crystals were soaked with a hydrolysable dCTP analog (dCMPP(CH2)P) with the hope of obtaining a structure resembling the product structure
Summary
Adenine frequently pairs with the Hoogsteen edge of an oxidized guanine base (8OG) causing G to T transversions. To examine the structural basis of this reduced efficiency, we sought to obtain the structure of the “product” complex of DNA polymerase (pol) β with the (syn)8OG:dA base pair at the primer terminus by soaking the binary complex crystals with a hydrolysable dCTP analogue complementary to the template base G. Crystallographic refinement of the structure revealed that the adenine of the (syn)8OG:dA base pair had been expelled from the primer terminus and a dCMP was inserted opposite 8OG in a reverse orientation; another uninserted molecule of the analogue was bound to the templating base G. This leads to an abortive complex that could form the basis of oxidatively-induced pol β stalling. The 8OG:dA base pair is readily extended by most polymerases leading to G to T transversions, as the 8OG:dA base pair is not recognized as a lesion by the mismatch repair machinery or exonucleases; this is because of resemblance of the 8OG:dA base pair hydrogen bonding to that of a dT:dA base pair
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