The Steric Gate Amino Acid Tyrosine 112 Is Required for Efficient Mismatched-Primer Extension by Human DNA Polymerase κ

Abstract

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 kappa (hPolkappa) is a member of the Y-family of DNA Pols and a direct counterpart of DinB in Escherichia coli. hPolkappa is characterized by its ability to bypass several DNA adducts [e.g., benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) and thymine glycol] and efficiently extend primers with mismatches at the termini. hPolkappa is structurally distinct from E. coli DinB in that it possesses an approximately 100-amino acid extension at the N-terminus. Here, we report that tyrosine 112 (Y112), the steric gate amino acid of hPolkappa, 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., k(cat), of the extending mismatched primers and lowered the efficiency, i.e., k(cat)/K(m), of this process by approximately 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-N(2)-dG in template DNA, nor did it affect the ability of hPolkappa to bind strongly to template-primer DNA with BPDE-N(2)-dG/dCMP. We conclude that the steric gate of hPolkappa is a major fidelity factor that regulates extension reactions from mismatched primer termini.

Impact of Ribonucleotide Backbone on Translesion Synthesis and Repair of 7,8-Dihydro-8-oxoguanine

“…Human pol ␣ was purchased from CHIMERx (WI). Human pol was purified as C-terminal truncations with 10 His tags as previously described (49). Human pol was purchased from Enzy- OGG1 (750 nM).…”

Variants of mouse DNA polymerase κ reveal a mechanism of efficient and accurate translesion synthesis past a benzo[ a ]pyrene dG adduct

“…Previous studies have investigated the fidelity of Y-family polymerases by mutating the steric gate and residues surrounding the incoming nucleotide, e.g., Phe13 and Tyr79 of DinB (15,27), Phe12 in Dbh (28), Tyr112 of Polκ (29), and Tyr11 of UmuC (30,31). Similarly, TLS efficiency and specificity have been examined after swapping the LF domain (32), the finger domain (33), or the linker between the thumb and LF among Y-family members (34).…”

In vivo evidence that DNA polymerase kappa is responsible for error-free bypass across DNA cross-links induced by mitomycin C

“…Polk is unique in that its orthologs are present in Eukarya, bacteria and Archaea [15][16][17]. Several lines of in vitro evidence suggest that Polk may be involved in TLS across a variety of DNA damage, such as N 2 -guanyl adducts induced by polycyclic aromatic hydrocarbons and alkylating agents [18][19][20][21][22][23], a C8-guanyl adduct by 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) [24], thymine glycol [25,26], 8-oxo-guanine [27,28], and interstrand DNA cross-links [29]. In addition to TLS, Polk is reported to be involved in nucleotide excision repair [30], replication checkpoint [31], repair of single-strand breaks in DNA [32], and microsatellite stability [33].…”