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Repair of DNA double-strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to ∼1–2 molecules per DNA end. The mechanisms enforcing this limit are currently unclear. Here, we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process that actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism that operates in S phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together, our data shed light on the multiple mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions.

The activities of many DNA-repair proteins are controlled through reversible covalent modification by ubiquitin and ubiquitin-like molecules. Nonhomologous end-joining (NHEJ) is the predominant DNA double-strand break (DSB) repair pathway in mammalian cells and is initiated by DSB ends being recognized by the Ku70/Ku80 (Ku) heterodimer. By using MLN4924, an anti-cancer drug in clinical trials that specifically inhibits conjugation of the ubiquitin-like protein, NEDD8, to target proteins, we demonstrate that NEDD8 accumulation at DNA-damage sites is a highly dynamic process. In addition, we show that depleting cells of the NEDD8 E2-conjugating enzyme, UBE2M, yields ionizing radiation hypersensitivity and reduced cell survival following NHEJ. Finally, we demonstrate that neddylation promotes Ku ubiquitylation after DNA damage and release of Ku and Ku-associated proteins from damage sites following repair. These studies provide insights into how the NHEJ core complex dissociates from repair sites and highlight its importance for cell survival following DSB induction.

Aiming to identify novel phosphorylation sites in response to DNA double-strand breaks (DSB) inducers, we have isolated a phosphorylation site on KU70. Unexpectedly, a rabbit antiserum raised against this site cross-reacted with a 120 kDa protein in cells treated by DNA DSB inducers. We identified this protein as SAF-A/hnRNP U, an abundant and essential nuclear protein containing regions binding DNA or RNA. The phosphorylation site was mapped at S59 position in a sequence context favoring a "S-hydrophobic" consensus model for DNA-PK phosphorylation site in vivo. This site was exclusively phosphorylated by DNA-PK in response to DNA DSB inducers. In addition, the extent and duration of this phosphorylation was in inverse correlation with the capacity of the cells to repair DSB by nonhomologous end joining. These results bring a new link between the hnRNP family and the DNA damage response. Additionaly, the mapped phospho-site on SAF-A might serve as a potential bio-marker for DNA-PK activity in academic studies and clinical analyses of DNA-PK activators or inhibitors.

The c-MYC proto-oncogene encodes a transcription factor that is critical for cell growth and proliferation. It is one of the genes frequently altered in cancer cells in which it exhibits constitutive activity. The half-life of c-MYC is very short in quiescent cells due to ubiquitin-mediated proteolysis. We report here the rapid and dose-dependent decline of c-MYC protein level after UV-irradiation in various human and rodent cells. This decline is due to a proteasomal degradation of c-MYC protein and does not require the binding sites for the FBW7 and SKP2 ubiquitin ligases. Together, our data exclude a prominent role for the stress-responsive kinase PAK2, for the major phosphoinositide 3-kinase related protein kinases ATR, ATM, DNA-PK and mTOR and for ERK, JNK and p38 mitogen activated protein kinases in this UV-induced degradation process. We propose that c-MYC degradation is part of the global cell response to UV-damage, complementary to the accumulation and activation of the p53 transcription factor. By contributing to the replication arrest after infliction of lesions to the genome, the induced degradation of c-MYC may be part of the safeguard mechanisms maintaining genome stability.

Association of the DNA end-binding Ku70/Ku80 heterodimer with the 460-kDa serine/threonine kinase catalytic subunit forms the DNA-dependent protein kinase (DNA-PK) that is required for double-strand break repair by non-homologous recombination in mammalian cells. Recently, we have proposed a model in which the kinase activity is required for translocation of the DNA end-binding subunit Ku along the DNA helix when DNA-PK assembles on DNA ends. Here, we have questioned the consequences of Ku entry into DNA on local DNA processes by using human nuclear cell extracts incubated in the presence of linearized plasmid DNA. As two model processes, we have chosen nucleotide excision repair (NER) of UVC DNA lesions and transcription from viral promoters. We show that although NER efficiency is strongly reduced on linear DNA, it can be fully restored in the presence of DNA-PK inhibitors. Simultaneously, the amount of NER proteins bound to the UVC-damaged linear DNA is increased and the amount of Ku bound to the same DNA molecules is decreased. Similarly, the poor transcription efficiency exhibited by viral promoters on linear DNA is enhanced in the presence of DNA-PK inhibitor concentrations that prevent Ku entry into the DNA substrate molecule. The present results show that DNA-PK catalytic activity can regulate DNA transactions including transcription in the vicinity of double-strand breaks by controlling Ku entry into DNA.

The nucleotide excision repair pathway contributes to genetic stability by removing a wide range of DNA damage through an error‐free reaction. When the lesion is located, the altered strand is incised on both sides of the lesion and a damaged oligonucleotide excised. A repair patch is then synthesized and the repaired strand is ligated. It is assumed that only DNA polymerases δ and/or ε participate to the repair DNA synthesis step. Using UV and cisplatin‐modified DNA templates, we measured in vitrothat extracts from cells overexpressing the error‐prone DNA polymerase β exhibited a five‐to sixfold increase of the ultimate DNA synthesis activity compared with control extracts and demonstrated the specific involvement of Pol β in this step. By using a 28 nt gapped, double‐stranded DNA substrate mimicking the product of the incision step, we showed that Pol β is able to catalyze strand displacement downstream of the gap. We discuss these data within the scope of a hypothesis previously presented proposing that excess error‐prone Pol β in cancer cells could perturb the well‐defined specific functions of DNA polymerases during error‐free DNA transactions.—Canitrot, Y., Hoffmann, J.‐S., Calsou, P., Hayakawa, H., Salles, B., Cazaux, C. Nucleotide excision repair DNA synthesis by excess DNA polymerase β: a potential source of genetic instability in cancer cells. FASEB J.14, 1765–1774 (2000)

ABSTRACTExposure of mammalian cells to short-wavelength light (UVC) triggers a global response which can either counteract the deleterious effect of DNA damage by enabling DNA repair or lead to apoptosis. Several stress-activated protein kinases participate in this response, making phosphorylation a strong candidate for being involved in regulating the cellular damage response. One factor that is phosphorylated in a UVC-dependent manner is the 32-kDa subunit of the single-stranded DNA-binding replication protein A (RPA32). RPA is required for major cellular processes like DNA replication, and removal of DNA damage by nucleotide excision repair (NER). In this study we examined the signal which triggers RPA32 hyperphosphorylation following UVC irradiation in human cells. Hyperphosphorylation of RPA was observed in cells from patients with either NER or transcription-coupled repair (TCR) deficiency (A, C, and G complementation groups of xeroderma pigmentosum and A and B groups of Cockayne syndrome, respectively). This exclude both NER intermediates and TCR as essential signals for RPA hyperphosphorylation. However, we have observed that UV-sensitive cells deficient in NER and TCR require lower doses of UV irradiation to induce RPA32 hyperphosphorylation than normal cells, indicating that persistent unrepaired lesions contribute to RPA phosphorylation. Finally, the results of UVC irradiation experiments on nonreplicating cells and S-phase-synchronized cells emphasize a major role for DNA replication arrest in the presence of UVC lesions in RPA UVC-induced hyperphosphorylation in mammalian cells.

Reactive oxygen species (ROS) damage DNA which appears to represent the major target involved in mutagenesis, carcinogenesis, and aging cell responses. Various DNA modifications are generated by ROS, but 8-hydroxy-2′-deoxyguanosine (8-oxoG) has retained a lot of attention in the last few years. Therefore, numerous methods have been developed to detect and quantify the extent of 8-oxoG in DNA, most of them requiring a significant amount of DNA that might be limiting in the case of biological samples. 8-oxoG is repaired in Escherichia coli by a specific glycosylase, the Fpg (formamidopyrimidine DNA glycosylase) protein, in a reaction that requires a covalent intermediate favored under reducing conditions. We set up a new assay based on the capture of plasmid DNA into sensitized microplate wells. DNA damaged by photoactivation of methylene blue was adsorbed on a polylysine-treated plastic well. Then the Fpg protein was added, allowed to fix on the damage by taking advantage of minimized glycosylase activity at low temperature and the reductive trapping of the covalent intermediate, yielding to a stable DNA–protein interaction. The trapped protein was subsequently recognized by a specific antibody. A secondary antibody coupled with horseradish peroxidase was used to detect the complex and the measurement was carried out by chemiluminescence. This new assay offers various potentialities, specifically in the field of technology of ROS producers.