Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1.

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein-interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku(-/-) mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847.

Translesion polymerase η is upregulated by cancer therapeutics and confers anticancer drug resistance.

DNA repair processes are a key determinant of the sensitivity of cancer cells to DNA-damaging chemotherapeutics, which may induce certain repair genes as a mechanism to promote resistance. Here, we report the results of a screen for repair genes induced in cancer cells treated with DNA crosslinking agents, which identified the translesion polymerase η (PolH) as a p53-regulated target acting as one defense against interstrand crosslink (ICL)-inducing agents. PolH was induced by fotemustine, mafosfamide, and lomustine in breast cancer, glioma, and melanoma cells in vitro and in vivo, with similar inductions observed in normal cells such as lymphocytes and diploid fibroblasts. PolH contributions to the protection against ICL-inducing agents were evaluated by its siRNA-mediated attenuation in cells, which elevated sensitivity to these drugs in all tumor cell models. Conversely, PolH overexpression protected cancer cells against these drugs. PolH attenuation reduced repair of ICL lesions as measured by host cell reactivation assays and enhanced persistence of γH2AX foci. Moreover, we observed a strong accumulation of PolH in the nucleus of drug-treated cells along with direct binding to damaged DNA. Taken together, our findings implicated PolH in ICL repair as a mechanism of cancer drug resistance and normal tissue protection.

Inefficient double-strand break repair in murine rod photoreceptors with inverted heterochromatin organization.

BACKGROUND: DNA double-strand break (DSB) repair is crucial for the maintenance of genomic stability, and chromatin organization represents one important factor influencing repair efficiency. Mouse rod photoreceptors with their inverted heterochromatin organization containing a single large chromocenter in the middle of the nucleus provide a unique model system to study DSB repair in heterochromatin of living animals. RESULTS: We observed that adult rod photoreceptors repair only half of the induced DSBs within 1 day after damage induction, a defect that is neither observed in any other cell type of the adult retina nor in rod photoreceptor precursor cells of postnatal day 4 mice. We show that adult wild-type rods are deficient in a repair pathway involving ATM, a protein that promotes heterochromatic DSB repair by phosphorylating KAP1 and facilitating heterochromatin relaxation. Of note, we observed that rods fail to robustly accumulate active ATM at DSBs, exhibit low KAP1 levels, and display high levels of SPOC1, a factor suppressing KAP1 phosphorylation. Collectively, this results in dramatically reduced KAP1 phosphorylation and the inability to repair heterochromatic DSBs. CONCLUSIONS: Because the distinct heterochromatic structure of rods focuses transmitting light to enable vision at low photon levels, the inability to phosphorylate KAP1 and the failure to relax heterochromatin could serve to maintain this structure and the functionality of rods in the presence of DSBs. Collectively, our findings show that the unique chromatin organization of adult rods renders them incapable to efficiently repair heterochromatic DSBs, providing evidence that heterochromatin affects mammalian DSB repair in vivo.

Near-infrared exposure changes cellular responses to ionizing radiation.

Near infrared (NIR) and X-rays are radiations from different sides of the wavelength spectrum but both are used during medical treatments, as they have severe impacts on cellular processes, including metabolism, gene expression, proliferation and survival. However, both radiations differ strictly in their consequences for exposed patients: NIR effects are generally supposed to be positive, mostly ascribed to a stimulation of metabolism, whereas X-ray leads to genetic instability, an increase of reactive oxygen species (ROS) and DNA damages and finally to cellular death by apoptosis in tumor cells. Since genomic stability after X-irradiation depends on the mitochondrial metabolism, which is well known to be regulated by NIR, we analyzed the impact of NIR on cellular responses of fibroblasts, retinal progenitor cells and keratinocytes to X-radiation. Our data show that previous exposure to naturally occurring doses of nonthermal NIR combined with clinically relevant X-ray doses leads to (1) increased genomic instability, indicated by elevated ratios of mitotic catastrophes, (2) increased ROS, (3) higher amounts of X-irradiated cells entering S-phase and (4) impaired DNA double-strand break repair. Taken together, our data show tremendous effects of NIR on cellular responses to X-rays, probably affecting the results of radiotherapy after NIR exposure during cancer treatment.

Brca2/Xrcc2 dependent HR, but not NHEJ, is required for protection against O(6)-methylguanine triggered apoptosis, DSBs and chromosomal aberrations by a process leading to SCEs.

O(6)-methylguanine (O(6)MeG) is a highly critical DNA adduct induced by methylating carcinogens and anticancer drugs such as temozolomide, streptozotocine, procarbazine and dacarbazine. Induction of cell death by O(6)MeG lesions requires mismatch repair (MMR) and cell proliferation and is thought to be dependent on the formation of DNA double-strand breaks (DSBs) or, according to an alternative hypothesis, direct signaling by the MMR complex. Given a role for DSBs in this process, either homologous recombination (HR) or non-homologous end joining (NHEJ) or both might protect against O(6)MeG. Here, we compared the response of cells mutated in HR and NHEJ proteins to temozolomide and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The data show that cells defective in HR (Xrcc2 and Brca2 mutants) are extremely sensitive to cell death by apoptosis and chromosomal aberration formation and less sensitive to sister-chromatid exchange (SCE) induction than the corresponding wild-type. Cells defective in NHEJ were not (Ku80 mutant), or only slightly more sensitive (DNA-PK(cs) mutant) to cell death and showed similar aberration and SCE frequencies than the corresponding wild-type. Transfection of O(6)-methylguanine-DNA methyltransferase (MGMT) in all of the mutants almost completely abrogated the genotoxic effects in both HR and NHEJ defective cells, indicating the mutant-specific hypersensitivity was due to O(6)MeG lesions. MNNG provoked H2AX phosphorylation 24-48h after methylation both in wild-type and HR mutants, which was not found in MGMT transfected cells. The gammaH2AX foci formed in response to O(6)MeG declined later in wild-type but not in HR-defective cells. The data support a model where DSBs are formed in response to O(6)MeG in the post-treatment cell cycle, which are repaired by HR, but not NHEJ, in a process that leads to SCEs. Therefore, HR can be considered as a mechanism that causes tolerance of O(6)MeG adducts. The data implicate that down-regulation or inhibition of HR might be a powerful strategy in improving cancer therapy with methylating agents.