Differing conformational pathways before and after chemistry for insertion of dATP versus dCTP opposite 8-oxoG in DNA polymerase beta

Abstract

To elucidate how human DNA polymerase beta (pol beta) discriminates dATP from dCTP when processing 8-oxoguanine (8-oxoG), we analyze a series of dynamics simulations before and after the chemical step with dATP and dCTP opposite an 8-oxoG template started from partially open complexes of pol beta. Analyses reveal that the thumb closing of pol beta before chemistry is hampered when the incorrect nucleotide dATP is bound opposite 8-oxoG; the unfavorable interaction between active-site residue Tyr(271) and dATP that causes an anti to syn change in the 8-oxoG (syn):dATP complex explains this slow motion, in contrast to the 8-oxoG (anti):dCTP system. Such differences in conformational pathways before chemistry for mismatched versus matched complexes help explain the preference for correct insertion across 8-oxoG by pol beta. Together with reference studies with a nonlesioned G template, we propose that 8-oxoG leads to lower efficiency in pol beta's incorporation of dCTP compared with G by affecting the requisite active-site geometry for the chemical reaction before chemistry. Furthermore, because the active site is far from ready for the chemical reaction after partial closing or even full thumb closing, we suggest that pol beta is tightly controlled not only by the chemical step but also by a closely related requirement for subtle active-site rearrangements after thumb movement but before chemistry.

FIGURE 1

The molecular formulas of guanine (a) and 8-oxoG (b) and the two possible 8-oxoG pairing forms: (c) anti Watson-Crick pairing, with C; and (d) syn Hoogsteen pairing, with A.

FIGURE 2

Top and side views of α-helices N in the simulated pol β complexes before (top) and after (bottom) the chemical reaction compared with those in the binary and ternary crystallographic structures of pol β (19). Pol β/substrate complexes with the 8-oxoG (anti):dC(TP), 8-oxoG (syn):dA(TP), and 8-oxoG (anti):dA(TP) nascent basepairs are shown in blue, purple, and gray, respectively. Superimposition is performed according to the palm subdomains.

FIGURE 3

Final conformations of the active-site residues in the simulated complexes before (top) and after (bottom) the chemical reaction compared with those in the binary gapped and ternary crystallographic structures. Superimposition is done according to the palm subdomains. The residue side chains and protein backbones in the complexes with 8-oxoG (anti):dC(TP), 8-oxoG (syn):dA(TP), and 8-oxoG (anti):dA(TP) are colored blue, purple, and gray, respectively. The magnesium ions are shown by green spheres.

FIGURE 4

The final conformations of the nascent basepairs in the simulated complexes before and after the chemical reaction compared with those of the ternary (red) and binary nicked (green) crystallographic structures. Color schemes for the three complexes are as labeled.

FIGURE 5

Interaction energies between the nascent basepair and Tyr²⁷¹ in the simulations before chemistry. Red and blue lines represent the total interaction energies between Tyr²⁷¹ and dATP or dCTP, and between Tyr²⁷¹ and the template 8-oxoG, respectively.

FIGURE 6

(a) Time evolution of the N-glycosidic bond torsion angle of dATP. (b) The initial (atoms in cyan, blue, red, and orange are C, N, O, and P, respectively) and final (purple) conformations of the nascent basepair in the 8-oxoG (syn):dATP pol β complex. Superimposition is performed according to the palm subdomain.