Significance: The endogenous genome damage induced by mitochondrial/cytosolic reactive oxygen species (ROS) is well recognized. However, similar damage induced by nuclear ROS generated via histone/cytosine-phosphate-guanine (CpG) demethylation during transcription has not been scrupulously investigated. This report documents the formation of genomic oxidized bases and single-strand breaks during ligand-induced gene activation via histone/CpG demethylation. That repair of these damages occurs preferentially in promoters and is essential for transcriptional activation underscore the essentiality of promoter-specific repair for transcription. In contrast, heat shock (HS) induction generates double-strand breaks, the repair of which is essential for the activation of HS-responsive genes. This study thus implies gross underestimation of endogenous oxidative genome damage and highlights the intrinsic diversity of damage and distinct repair processes associated with transcription.

Polynucleotide kinase/phosphatase (PNKP) has been implicated in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). To assess the consequences of PNKP deficiency for NHEJ of 3′-phosphate-ended DSBs, PNKP-deficient derivatives of HCT116 and of HeLa cells were generated using CRISPR/CAS9. For both cell lines, PNKP deficiency conferred sensitivity to ionizing radiation as well as to neocarzinostatin (NCS), which specifically induces DSBs bearing protruding 3′-phosphate termini. Moreover, NCS-induced DSBs, detected as 53BP1 foci, were more persistent in PNKP ^−/− HCT116 cells compared to their wild-type (WT) counterparts. Surprisingly, PNKP-deficient whole-cell and nuclear extracts were biochemically competent in removing both protruding and recessed 3′-phosphates from synthetic DSB substrates, albeit much less efficiently than WT extracts, suggesting an alternative 3′-phosphatase. Measurements by ligation-mediated PCR showed that PNKP-deficient HeLa cells contained significantly more 3′-phosphate-terminated and fewer 3′-hydroxyl-terminated DSBs than parental cells 5–15 min after NCS treatment, but this difference disappeared by 1 h. These results suggest that, despite presence of an alternative 3′-phosphatase, loss of PNKP significantly sensitizes cells to 3′-phosphate-terminated DSBs, due to a 3′-dephosphorylation defect.

Posttranslational modifications of DNA repair proteins have been linked to their function. However, it is not clear if posttranslational acetylation affects subcellular localization of these enzymes. Here, we show that the human DNA glycosylase NEIL1, which is involved in repair of both endo- and exogenously generated oxidized bases via the base excision repair (BER) pathway, is acetylated by histone acetyltransferase p300. Acetylation occurs predominantly at Lys residues 296, 297 and 298 located in NEIL1’s disordered C-terminal domain. NEIL1 mutant having the substitution of Lys 296–298 with neutral Ala lost nuclear localization, whereas Lys > Arg substitution (in 3KR mutant) at the same sites did not affect NEIL1’s nuclear localization or chromatin binding, presumably due to retention of the positive charge. Although non-acetylated NEIL1 can bind to chromatin, acetylated NEIL1 is exclusively chromatin-bound. NEIL1 acetylation while dispensable for its glycosylase activity enhances it due to increased product release. The acetylation-defective 3KR mutant forms less stable complexes with various chromatin proteins, including histone chaperones and BER/single-strand break repair partners, than the wild-type (WT) NEIL1. We also showed that the repair complex with WT NEIL1 has significantly higher BER activity than the 3KR mutant complex. This is consistent with reduced resistance of non-acetylable mutant NEIL1 expressing cells to oxidative stress relative to cells expressing acetylable WT enzyme. We thus conclude that the major role of acetylable Lys residues in NEIL1 is to stabilize the formation of chromatin-bound repair complexes which protect cells from oxidative stress.

There is increasing interest in developing and applying DNA repair inhibitors in cancer treatment to augment the efficacy of radiation and conventional genotoxic chemotherapy. However, targeting the inhibitor is required to avoid reducing the repair capacity of normal tissue. The aim of this study was to develop nano-delivery systems for the encapsulation of novel imidopiperidine-based inhibitors of the DNA 3′-phosphatase activity of polynucleotide kinase/phosphatase (PNKP), a DNA repair enzyme that plays a critical role in rejoining DNA single- and double-strand breaks. For this purpose, newly identified hit compounds with potent PNKP inhibitory activity, A12B4C50 and A83B4C63, were encapsulated in polymeric micelles of different poly(ethylene oxide)-b-poly(ɛ-caprolactone) (PEO-b-PCL)-based structures. Our results showed efficient loading of A12B4C50 and A83B4C63 in PEO-b-PCLs with pendent carboxyl and benzyl carboxylate groups, respectively, and relatively slow release over 24 h. Both free and encapsulated inhibitors were able to sensitize HCT116 cells to radiation and the topoisomerase I poison, irinotecan. In addition, the encapsulated inhibitors were capable of inducing synthetic lethalilty in Phosphatase and tensin homolog (PTEN)-deficient cells. We also established the validity of the peptide GE11 as a suitable ligand for active targeted delivery of nano-encapsulated drugs to colorectal cancer cells overexpressing epidermal growth factor receptor (EGFR). Our results show the potential of nano-encapsulated inhibitors of PNKP as either mono or combined therapeutic agents for colorectal cancer.

We have developed a label-free, simple and highly sensitive hairpin fluorescent biosensor for the assay of DNA 3′-phosphatases and their inhibitors utilizing a graphene oxide (GO) platform. In this assay, we designed a hairpin primer (HP) with a 3′-phosphoryl end that served as the substrate for DNA 3′-phosphatases. Once the phosphorylated HP was hydrolyzed by DNA 3′-phosphatases, the resulting HP with a 3′-hydroxyl end was immediately elongated to form a long double-strand product by Klenow fragment polymerase (KF polymerase). With SYBR green I (SG) selective staining of the double-helix DNA, a very high fluorescence enhancement was achieved. Furthermore, GO was introduced to quench the fluorescence of the HP without polymerase elongation, thereby further increasing the signal-to-background ratio. The proposed method is simple and convenient, yet still exhibits high sensitivity and selectivity. This method has been successfully applied to detecting the activity of two typical 3′-phosphatases, T4 polynucleotide kinase phosphatase (PNKP) and shrimp alkaline phosphatase (SAP). The effect of their inhibitors has also been investigated. The results revealed that the method allowed a sensitive quantitative assay of T4 PNKP and SAP, with detection limits of 0.07 U mL⁻¹ and 0.003 U mL⁻¹, respectively. The proposed method is anticipated to find applications in the study of DNA damage repair mechanisms.

A genetic screen using a library of 6,961 siRNAs led to the identification of SHP-1 (PTPN6), a tumor suppressor frequently mutated in malignant lymphomas, leukemias, and prostate cancer, as a potential synthetic lethal partner of the DNA repair protein polynucleotide kinase/phosphatase (PNKP). After confirming the partnership with SHP-1, we observed that codepletion of PNKP and SHP-1 induced apoptosis. A T-cell lymphoma cell line that is SHP-1 deficient (Karpas 299) was shown to be sensitive to a chemical inhibitor of PNKP, but resistance was restored by expression of wild-type SHP-1 in these cells. We determined that while SHP-1 depletion does not significantly impact DNA strand-break repair, it does amplify the level of reactive oxygen species (ROS) and elevate endogenous DNA damage. The ROS scavenger WR1065 afforded protection to SHP-1–depleted cells treated with the PNKP inhibitor. We propose that codisruption of SHP-1 and PNKP leads to an increase in DNA damage that escapes repair, resulting in the accumulation of cytotoxic double-strand breaks and induction of apoptosis. This supports an alternative paradigm for synthetic lethal partnerships that could be exploited therapeutically. Cancer Res; 72(22); 5934–44. ©2012 AACR.

Mutations in mitochondrial DNA (mtDNA) are implicated in a broad range of human diseases and in aging. Compared to nuclear DNA, mtDNA is more highly exposed to oxidative damage due to its proximity to the respiratory chain and the lack of protection afforded by chromatin-associated proteins. While repair of oxidative damage to the bases in mtDNA through the base excision repair pathway has been well studied, the repair of oxidatively induced strand breaks in mtDNA has been less thoroughly examined. Polynucleotide kinase/phosphatase (PNKP) processes strand-break termini to render them chemically compatible for the subsequent action of DNA polymerases and ligases. Here, we demonstrate that functionally active full-length PNKP is present in mitochondria as well as nuclei. Downregulation of PNKP results in an accumulation of strand breaks in mtDNA of hydrogen peroxide-treated cells. Full restoration of repair of the H ₂ O ₂ -induced strand breaks in mitochondria requires both the kinase and phosphatase activities of PNKP. We also demonstrate that PNKP contains a mitochondrial-targeting signal close to the C-terminus of the protein. We further show that PNKP associates with the mitochondrial protein mitofilin. Interaction with mitofilin may serve to translocate PNKP into mitochondria.

DNA 3′‐phosphatases play a unique role in the repair of strand breaks induced by DNA damaging agents, such as ionizing radiation or oxidative stress. In this paper, we present an efficient detection system for rapid screening of DNA 3′‐phosphatases and their inhibitors. A unique template substrate has been designed to hybridize with the universal molecular beacon (U‐MB), and the detection process is carried out in a quantitative real‐time PCR. The method is successfully applied to monitor the activity and kinetics of two typical 3′‐phosphatases, that is, T4 polynucleotide kinase phosphatase (PNKP) and calf intestinal alkaline phosphatase (CIP). The inhibition effect of heparin on T4 PNKP and theophylline on CIP is also quantitatively characterized. The proposed method is demonstrated to be very useful for sensitive, high‐throughput, and precise measurement of various 3′‐phosphatases and their inhibitors.

XRCC1 plays a central role in mammalian single-strand break repair. Although it has no enzymatic activity of its own, it stimulates the activities of polynucleotide kinase/phosphatase (PNKP), and this function is enhanced by protein kinase CK2 mediated phosphorylation of XRCC1. We have previously shown that non-phosphorylated XRCC1 stimulates the kinase activity of PNKP by increasing the turnover of PNKP. Here we extend our analysis of the XRCC1-PNKP interaction taking into account the phosphorylation of XRCC1. We demonstrate that phosphorylated and non-phosphorylated XRCC1 interact with different regions of PNKP. Phosphorylated XRCC1 binds with high affinity ( K_d = 3.5 nM and 1 : 1 stoichiometry) to the forkhead associated (FHA) domain, while non-phosphorylated XRCC1 binds to the catalytic domain of PNKP with lower affinity ( K_d = 43.0 nM and 1 : 1 stoichiometry). Under conditions of limited enzyme concentration both forms of XRCC1 enhance the activities of PNKP, but the effect is more pronounced with phosphorylated XRCC1, particularly for the kinase activity of PNKP. The stimulatory effect of phosphorylated XRCC1 on PNKP can be totally inhibited by the presence of excess FHA domain polypeptide, but non-phosphorylated XRCC1 is not susceptible to competition by the FHA domain. Thus, XRCC1 can stimulate PNKP by two independent mechanisms.

Human polynucleotide kinase (hPNK) is a bifunctional enzyme possessing a 5′-DNA kinase activity and a 3′-phosphatase activity. Studies based on cell extracts and purified proteins have indicated that hPNK can act on single-strand breaks and double-strand breaks (DSB) to restore the termini to the chemical form required for further action by DNA repair polymerases and ligases (i.e., 5′-phosphate and 3′-hydroxyl termini). These studies have revealed that hPNK can bind to XRCC4, and as a result, hPNK has been implicated as a participant in the nonhomologous end joining (NHEJ) pathway for DSB repair. We sought to confirm the role of hPNK in NHEJ in the cellular setting using a genetic approach. hPNK was stably down-regulated by RNA interference expression in M059K glioblastoma cells, which are NHEJ positive, and M059J cells, which are NHEJ deficient due to a lack of DNA-PK catalytic subunit (DNA-PKcs). Whereas depletion of hPNK significantly sensitized M059K cells to ionizing radiation, no additional sensitization was conferred to M059J cells, clearly implying that hPNK operates in the same DNA repair pathway as DNA-PKcs. On the other hand, depletion of hPNK did not increase the level of sister chromatid exchanges, indicating that hPNK is not involved in the homologous recombination DSB repair pathway. We also provide evidence that the action of hPNK in the repair of camptothecin-induced topoisomerase 1 “dead-end” complexes is independent of DNA-PKcs and that hPNK is not involved in the nucleotide excision repair pathway. [Cancer Res 2007;67(14):6619–25]

Ionizing radiation (IR) is an important environmental risk factor for various cancers and also a major therapeutic agent for cancer treatment. Exposure of mammalian cells to IR induces several types of damage to DNA, including double- and single-strand breaks, base and sugar damage, as well as DNA-DNA and DNA-protein cross-links (DPCs). Little is known regarding the biological consequences of DPCs. Identifying the proteins that become cross-linked to DNA by IR would be an important first step in this regard. We have therefore undertaken a proteomics study to isolate and identify proteins involved in IR-induced DPCs. DPCs were induced in AA8 Chinese hamster ovary or GM00637 human fibroblast cells using 0–4 gray of γ-rays under either aerated or hypoxic conditions. DPCs were isolated using a recently developed method, and proteins were identified by mass spectrometry. We identified 29 proteins as being cross-linked to DNA by IR under aerated and/or hypoxic conditions. The identified proteins include structural proteins, actin-associated proteins, transcription regulators, RNA-splicing components, stress-response proteins, cell cycle regulatory proteins, and GDP/GTP-binding proteins. The involvement of several proteins (actin, histone H2B, and others) in DPCs was confirmed by using Western blot analysis. The dose responsiveness of DPC induction was examined by staining one-dimensional SDS-polyacrylamide gels with SYPRO Tangerine followed by analysis using fluorescence imaging. Quantitation of the fluorescence signal indicated no significant difference in total yields of IR-induced DPCs generated under aerated or hypoxic conditions, although differences were observed for several individual protein bands.

We report the characterization of Pnk1, a 45-kDa homolog of the human polynucleotide kinase PNKP inSchizosaccharomyces pombe. Recombinant Pnk1 like human PNKP exhibits both 5′-DNA kinase and 3′-DNA phosphatase activities in vitro. Furthermore, we detected 3′-DNA phosphatase activity with a single-stranded substrate in extracts from wild-type yeast, but no activity was detected in pnk1Δ strains. We have shown that GFP-tagged Pnk1 like mammalian PNKP localizes to the nucleus. Deletion of pnk1 does not affect cell growth under normal conditions but results in significant hypersensitivity to γ-radiation or camptothecin, an inhibitor of topoisomerase I, suggesting that Pnk1 plays an important role in the repair of DNA strand breaks produced by these agents. The pnk1 deletion mutants were not hypersensitive to ethyl methanesulfonate, methyl methanesulfonate, or 4-nitroquinoline N-oxide. Expression of human PNKP in pnk1Δ cells restores resistance to γ-radiation or camptothecin, suggesting that the functions of yeast Pnk1 and human PNKP have been conserved.