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., kcat, of the extending mismatched primers and lowered the efficiency, i.e., kcat/Km, 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. [ABSTRACT FROM AUTHOR]