2w9a

Publication Abstract from PubMed

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 N(2),N(2)-dimethyl (Me(2))-substituted guanine (N(2),N(2)-Me(2)G), in contrast to a single methyl substitution. Therefore, it is unclear why the addition of two methyl groups is so disruptive. The presence of N(2),N(2)-Me(2)G lowered the catalytic efficiency of the model enzyme Sulfolobus solfataricus Dpo4 16,000-fold. Dpo4 inserted dNTPs almost at random during bypass of N(2),N(2)-Me(2)G, and much of the enzyme was kinetically trapped by an inactive ternary complex when N(2),N(2)-Me(2)G 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 N(2),N(2)-Me(2)G 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 N(2),N(2)-Me(2)G in which one hydrogen bond exists between the O-2 atom of C(dd) and the N-1 atom of N(2),N(2)-Me(2)G, with a second water-mediated hydrogen bond between the N-3 atom of C(dd) and the O-6 atom of N(2),N(2)-Me(2)G. A crystal structure of Dpo4 with dTTP opposite template N(2),N(2)-Me(2)G 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 N(2),N(2)-Me(2)G compared with mono-substituted N(2)-alkyl G adducts.

Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES.,Zhang H, Eoff RL, Kozekov ID, Rizzo CJ, Egli M, Guengerich FP J Biol Chem. 2009 Jun 26;284(26):17687-99. PMID:19542237^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.