The steric gate amino acid tyrosine 112 is required for efficient mismatched-primer extension by human DNA polymerase κ

Human DNA is continuously damaged by exogenous and endogenous genotoxic insults. To counteract DNA damage and ensure the completion of DNA replication, cells possess specialized DNA polymerases (Pols) that bypass a variety of DNA lesions. Human DNA polymerase κ (hPolκ) is a member of the Y-family of DNA Pols and a direct counterpart of DinB in Escherichia coli. hPolκ is characterized by its ability to bypass several DNA adducts [e.g., benzo[a]pyrene diolepoxide-N²-deoxyguanine (BPDE-N²-dG) and thymine glycol] and efficiently extend primers with mismatches at the termini. hPolκ is structurally distinct from E. coli DinB in that it possesses an ∼100-amino acid extension at the N-terminus. Here, we report that tyrosine 112 (Y112), the steric gate amino acid of hPolκ, which distinguishes dNTPs from rNTPs by sensing the 2′-hydroxy group of incoming nucleotides, plays a crucial role in extension reactions with mismatched primer termini. When Y112 was replaced with alanine, the amino acid change severely reduced the catalytic constant, i.e., κ_cat, of the extending mismatched primers and lowered the efficiency, i.e., κ_cat/ K_m, of this process by ∼400-fold compared with that of the wild-type enzyme. In contrast, the amino acid replacement did not reduce the insertion efficiency of dCMP opposite BPDE-N²-dG in template DNA, nor did it affect the ability of hPolκ to bind strongly to template-primer DNA with BPDE-N²-dG/dCMP. We conclude that the steric gate of hPolκ is a major fidelity factor that regulates extension reactions from mismatched primer termini.