Bypass of N2-ethylguanine by human DNA polymerase κ☆

Abstract

The efficiency and fidelity of nucleotide incorporation and next-base extension by DNA polymerase (pol) κ past N 2 -ethyl-Gua were measured using steady-state and rapid kinetic analyses. DNA pol κ incorporated nucleotides and extended 3′ termini opposite N 2 -ethyl-Gua with measured efficiencies and fidelities similar to that opposite Gua indicating a role for DNA pol κ at the insertion and extension steps of N 2 -ethyl-Gua bypass. The DNA pol κ was maximally activated to similar levels by a twenty-fold lower concentration of Mn 2+ compared to Mg 2+ . In addition, the steady state analysis indicated that high fidelity DNA pol κ-catalyzed N 2 -ethyl-Gua bypass is Mg 2+ -dependent. Strikingly, Mn 2+ activation of DNA pol κ resulted in a dramatically lower efficiency of correct nucleotide incorporation opposite both N 2 -ethyl-Gua and Gua compared to that detected upon Mg 2+ activation. This effect is largely governed by diminished correct nucleotide binding as indicated by the high K m values for dCTP insertion opposite N 2 -ethyl-Gua and Gua with Mn 2+ activation. A rapid kinetic analysis showed diminished burst amplitudes in the presence of Mn 2+ compared to Mg 2+ indicating that DNA pol κ preferentially utilizes Mg 2+ activation. These kinetic data support a DNA pol κ wobble base pairing mechanism for dCTP incorporation opposite N 2 -ethyl-Gua. Furthermore, the dramatically different polymerization efficiencies of the Y-family DNA pols κ and ι in the presence of Mn 2+ suggest a metal ion-dependent regulation in coordinating the activities of these DNA pols during translesion synthesis.

Elucidation of kinetic mechanisms of human translesionDNApolymerase κ using tryptophan mutants

“…For insertion of dCTP opposite unmodified G, wild-type and W214Y/W392H hpol j exhibited burst amplitudes of 68% and 64%, respectively, similar to a previous study with purified full-length hpol j and its catalytic core ( Fig. 3 and Table 3) [15,18,39]. Although mutants Y50W and T408W showed slightly lower burst amplitudes than wild-type hpol j (i.e.…”

Structural diversity of the Y-family DNA polymerases

“…Similarly, in a structure of pol ι with a smaller N 2 -ethyl-dG lesion at position 0, the base is found in a syn conformation, forming a Hoogsteen pair with an incoming dCTP, and the adduct is in the major groove above the DNA backbone [57]. In a final related structure, of Dpo4 with N 2 -dimethyl-dG template lesion at position 0, the damaged base shifts toward the minor groove, but remains in an anti conformation and forms a wobble pair with an incoming dTTP [58].…”

Acetaldehyde and the genome: Beyond nuclear DNA adducts and carcinogenesis

“…Based on studies with N 2 ‐ethyl‐dGuo, used as a structural model for N 2 ‐ethylidene‐dGuo, the adduct is only a transient block to the replicative DNA polymerase δ in vitro [Choi and Guengerich, ], However, in vivo studies using a double stranded lesion containing vector indicate that N 2 ‐ethyl‐dGuo is a significant block to replication, though only weakly mutagenic [Upton et al, ]. Cells have multiple specialized DNA polymerases that can bypass the adduct in a nonmutagenic manner (i.e., they insert dCMP opposite the lesion) [Choi et al, ; Choi and Guengerich, ; Pence et al, ].…”