Formation of primed single-stranded DNA at stalled replication forks triggers activation of the replication checkpoint signalling cascade resulting in the ATR-mediated phosphorylation of the Chk1 protein kinase, thus preventing genomic instability. By using siRNA-mediated depletion in human cells and immunodepletion and reconstitution experiments in Xenopus egg extracts, we report that the Y-family translesion (TLS) DNA polymerase kappa (Pol κ) contributes to the replication checkpoint response and is required for recovery after replication stress. We found that Pol κ is implicated in the synthesis of short DNA intermediates at stalled forks, facilitating the recruitment of the 9-1-1 checkpoint clamp. Furthermore, we show that Pol κ interacts with the Rad9 subunit of the 9-1-1 complex. Finally, we show that this novel checkpoint function of Pol κ is required for the maintenance of genomic stability and cell proliferation in unstressed human cells.

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.

DNA polymerase κ (Pol κ) is a specialized DNA polymerase involved in translesion DNA synthesis. Although its bypass activities across lesions are well characterized in biochemistry, its cellular protective roles against genotoxic insults are still elusive. To better understand the in vivo protective roles, we have established a human cell line deficient in the expression of Pol κ (KO) and another expressing catalytically dead Pol κ (CD), to examine the cytotoxic sensitivity to 11 genotoxins including ultraviolet C light (UV). These cell lines were established in a genetic background of Nalm‐6‐MSH+, a human lymphoblastic cell line that has high efficiency for gene targeting, and functional p53 and mismatch repair activities. We classified the genotoxins into four groups. Group 1 includes benzo[a]pyrene diolepoxide, mitomycin C, and bleomycin, where the sensitivity was equally higher in KO and CD than in the cell line expressing wild‐type Pol κ (WT). Group 2 includes hydrogen peroxide and menadione, where hypersensitivity was observed only in KO. Group 3 includes methyl methanesulfonate and ethyl methanesulfonate, where hypersensitivity was observed only in CD. Group 4 includes UV and three chemicals, where the chemicals exhibited similar cytotoxicity to all three cell lines. The results suggest that Pol κ not only protects cells from genotoxic DNA lesions via DNA polymerase activities, but also contributes to genome integrity by acting as a non‐catalytic protein against oxidative damage caused by hydrogen peroxide and menadione. The non‐catalytic roles of Pol κ in protection against oxidative damage by hydrogen peroxide are discussed. Environ. Mol. Mutagen. 56:650–662, 2015. © 2015 Wiley Periodicals, Inc.

In mammalian cells, DNA polymerase ␤ (Pol␤) functions in base excision repair. We have previously shown that Pol␤-deficient mice exhibit extensive neuronal cell death (apoptosis) in the developing nervous system and that the mice die immediately after birth. Here, we studied potential roles in the phenotype for p53, which has been implicated in DNA damage sensing, cell cycle arrest, and apoptosis. We generated Pol␤ ؊/؊ p53 ؊/؊ double-mutant mice and found that p53 deficiency dramatically rescued neuronal apoptosis associated with Pol␤ deficiency, indicating that p53 mediates the apoptotic process in the nervous system. Importantly, proliferation and early differentiation of neuronal progenitors in Pol␤ ؊/؊ p53 ؊/؊ mice appeared normal, but their brains obviously displayed cytoarchitectural abnormalities; moreover, the mice, like Pol␤mice, failed to survive after birth. Thus, we strongly suggest a crucial role for Pol␤ in the differentiation of specific neuronal cell types.Repair of DNA damage is essential for maintaining the integrity of the genetic information necessary for normal development and physiological consequences (28). DNA polymerase ␤ (Pol␤) is a 39-kDa protein of a single polypeptide, consisting of two catalytic, functional domains. The N-terminal 8-kDa domain carries a 5Ј-deoxyribose phosphate lyase activity, whereas the C-terminal 31-kDa domain carries a polymerase activity that fills a short gap with a 5Ј-phosphate (26,40). Pol␤ is a critical component of the base excision repair (BER) pathway. The BER pathway repairs DNA damage, such as apurinic/apyrimidinic (AP) sites and base modifications, which spontaneously occur or are induced by a variety of endogenous and exogenous agents, including reactive oxygen species and DNA alkylating agents. Biochemical studies have identified two types of BER in mammalian cells: a short-patch pathway involving replacement of one nucleotide and a long-patch pathway involving gap-filling of several nucleotides (46). The BER pathway is generally initiated by a specific DNA glycosylase that recognizes and removes a damaged base to generate an AP site in DNA, followed by incision of the site by an AP endonuclease. In the short-patch BER pathway, Pol␤ removes the 5Ј-deoxyribose phosphate and fills the single nucleotide gap, and finally DNA ligase I or a complex of XRCC1 and DNA ligase III ligates the nick. On the other hand, the long-