Distinct energetics and closing pathways for DNA polymerase beta with 8-oxoG template and different incoming nucleotides

Abstract

Background: 8-Oxoguanine (8-oxoG) is a common oxidative lesion frequently encountered by DNA polymerases such as the repair enzyme DNA polymerase beta (pol beta). To interpret in atomic and energetic detail how pol beta processes 8-oxoG, we apply transition path sampling to delineate closing pathways of pol beta 8-oxoG complexes with dCTP and dATP incoming nucleotides and compare the results to those of the nonlesioned G:dCTP and G:dATPanalogues.

Results: Our analyses show that the closing pathways of the 8-oxoG complexes are different from one another and from the nonlesioned analogues in terms of the individual transition states along each pathway, associated energies, and the stability of each pathway's closed state relative to the corresponding open state. In particular, the closed-to-open state stability difference in each system establishes a hierarchy of stability (from high to low) as G:C > 8-oxoG:C > 8-oxoG:A > G:A, corresponding to -3, -2, 2, 9 kBT, respectively. This hierarchy of closed state stability parallels the experimentally observed processing efficiencies for the four pairs. Network models based on the calculated rate constants in each pathway indicate that the closed species are more populated than the open species for 8-oxoG:dCTP, whereas the opposite is true for 8-oxoG:dATP.

Conclusion: These results suggest that the lower insertion efficiency (larger Km) for dATP compared to dCTP opposite 8-oxoG is caused by a less stable closed-form of pol beta, destabilized by unfavorable interactions between Tyr271 and the mispair. This stability of the closed vs. open form can also explain the higher insertion efficiency for 8-oxoG:dATP compared to the nonlesioned G:dATP pair, which also has a higher overall conformational barrier. Our study offers atomic details of the complexes at different states, in addition to helping interpret the different insertion efficiencies of dATP and dCTP opposite 8-oxoG and G.

Figure 1

The base pairing possibilities of 8-oxoG (8oG). In an anti conformation it forms a Watson-Crick base pair with dCTP (a); by assuming a syn conformation, it can form a Hoogsteen base pair with dATP.

Figure 2

Order parameter distributions in the 8-oxoG:dATP system. Normalized probability distributions of the order parameters of transition states 1 to 4 (TS1 to TS4) in the 8-oxoG:dATP complex. Labels 0 and 1 indicate the open and closed state conformations of the residues and thumb subdomain.

Figure 3

Order parameter distributions in the 8-oxoG:dCTP system. Normalized probability distributions of the order parameters in TS1 to TS4 in the 8-oxoG:dCTP complex. Labels 0 and 1 indicate the open and closed states of the residues and thumb subdomain.

Figure 4

The resolved free energy profiles. The free energy profiles of the 8-oxoG:dCTP and 8-oxoG:dATP complexes during pol β's thumb closing. The superimposed dashed lines are approximate free energy profiles of nonlesioned G:C (blue) and G:A (magenta) systems [32], respectively.

Figure 5

Simulation results based on the network models. The mean trajectories of the match (a) and mismatch (b) 8-oxoG systems obtained from 100 independent simulations with STOCKS [36]. The red, black, and green curves describe the evolution of the open, closed, and chemical product species, respectively.

Figure 6

Conformation of dATP in the 8-oxoG:dATP complex. The probability distributions of the N-glycosidic bond torsion angle of dATP during transition states 2 (Asp192 flip, blue), 3 (Arg258 rotation, red), and 4 (Phe272 flip, green) in the 8-oxoG:dATP complex.

Figure 7

The α-helix N and the nascent base pair conformations in the wild-type and mutant 8-oxoG:dATP complexes. The final conformations of α-helices N and the nascent base pair in the wild-type (magenta) and Y271A mutant (yellow) pol β complex simulations with 8-oxoG:dATP are superimposed according to their palm subdmains and compared to the crystal open (green) and closed (red) structures.