Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES*

Previous work has shown that Y-family DNA polymerases tolerate large DNA adducts, but a substantial decrease in catalytic efficiency and fidelity occurs during bypass of N²,N²-dimethyl (Me₂)-substituted guanine (N²,N²-Me₂G), in contrast to a single methyl substitution. Therefore, it is unclear why the addition of two methyl groups is so disruptive. The presence of N²,N²-Me₂G lowered the catalytic efficiency of the model enzyme Sulfolobus solfataricus Dpo4 16,000-fold. Dpo4 inserted dNTPs almost at random during bypass of N²,N²-Me₂G, and much of the enzyme was kinetically trapped by an inactive ternary complex when N²,N²-Me₂G was present, as judged by a reduced burst amplitude (5% of total enzyme) and kinetic modeling. One crystal structure of Dpo4 with a primer having a 3′-terminal dideoxycytosine (C_dd) opposite template N²,N²-Me₂G in a post-insertion position showed C_dd folded back into the minor groove, as a catalytically incompetent complex. A second crystal had two unique orientations for the primer terminal C_dd as follows: (i) flipped into the minor groove and (ii) a long pairing with N²,N²-Me₂G in which one hydrogen bond exists between the O-2 atom of C_dd and the N-1 atom of N²,N²-Me₂G, with a second water-mediated hydrogen bond between the N-3 atom of C_dd and the O-6 atom of N²,N²-Me₂G. A crystal structure of Dpo4 with dTTP opposite template N²,N²-Me₂G revealed a wobble orientation. Collectively, these results explain, in a detailed manner, the basis for the reduced efficiency and fidelity of Dpo4-catalyzed bypass of N²,N²-Me₂G compared with mono-substituted N²-alkyl G adducts.