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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nonhomologous end-joining (NHEJ) pathway of DNA double-strand break repair requires three protein complexes in Saccharomyces cerevisiae: MRX (Mre11-Rad50-Xrs2), Ku (Ku70-Ku80), and DNA ligase IV (Dnl4-Lif1-Nej1). Much is known about the interactions that mediate the formation of each complex, but little is known about how they act together during repair. A comprehensive yeast two-hybrid screen of the NHEJ factors of S. cerevisiae revealed all known interactions within the MRX, Ku, and DNA ligase IV complexes, as well as three additional, weaker interactions between Yku80-Dnl4, Xrs2-Lif1, and Mre11-Yku80. Individual and combined deletions of the Yku80 C terminus and the Xrs2 forkhead-associated (FHA) domain were designed based on the latter two-hybrid results. These deletions synergistically blocked NHEJ but not the telomere and recombination functions of Ku and MRX, confirming that these protein regions are functionally important specifically for NHEJ. Further mutational analysis of Yku80 identified a putative C-terminal amphipathic alpha-helix that is both required for its NHEJ function and strikingly similar to a DNA-dependent protein kinase interaction motif in human Ku80. These results identify a novel role in yeast NHEJ for the poorly characterized Ku80 C-terminal and Xrs2 FHA domains, and they suggest that redundant binding of DNA ligase IV facilitates completion of this DNA repair event.
Mol Cell Biol 2005 Dec
PMID:Mutations of the Yku80 C terminus and Xrs2 FHA domain specifically block yeast nonhomologous end joining. 1631 3

Two highly conserved double-strand break (DSB) repair pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ), function in all eukaryotes. How a cell chooses which pathway to utilize is an area of active research and debate. During NHEJ, the DNA-dependent protein kinase (DNA-PK) functions as a "gatekeeper" regulating DNA end access. Here, we provide evidence that DNA-PK regulates DNA end access via its own autophosphorylation. We demonstrated previously that autophosphorylation within a major cluster of sites likely mediates a conformational change that is critical for DNA end processing. Furthermore, blocking autophosphorylation at these sites inhibits a cell's ability to utilize the other major double-strand break repair pathway, HR. Here, we define a second major cluster of DNA-PK catalytic subunit autophosphorylation sites. Whereas blocking phosphorylation at the first cluster inhibits both end processing and HR, blocking phosphorylation at the second cluster enhances both. We conclude that separate DNA-PK autophosphorylation events may function reciprocally by not only regulating DNA end processing but also affecting DSB repair pathway choice.
Mol Cell Biol 2005 Dec
PMID:Autophosphorylation of DNA-dependent protein kinase regulates DNA end processing and may also alter double-strand break repair pathway choice. 1631 9

Although genotoxic agents are powerful inducers of stress kinases (SAPK/JNK), the contribution of DNA damage itself to this response is unknown. Therefore, SAPK/JNK activation of cells harboring specific defects in DNA damage-recognition mechanisms was studied. Dual phosphorylation of SAPK/JNK by the genotoxin methyl methanesulfonate (MMS) occurred in two waves. The early response (< or = 2 h after exposure) was similar in cells knockout for ATM, PARP, p53, and CSB or defective in DNA-PK(cs) compared with wild-type cells. The late response however (> or = 4 h), was drastically reduced in DNA-PK(cs) and Cockayne's syndrome B (CSB)-deficient cells. Similar results were obtained with human cells lacking DNA-PK(cs) and CSB. Activation of SAPK/JNK by MMS was not affected upon inhibition of base excision repair (BER), indicating base damage itself does not signal to SAPK/JNK. Because SAPK/JNK activation was attenuated in nongrowing cells, DNA replication-dependent processing of lesions, involving DNA-PK(cs) and CSB, appears to be required. DNA-PK(cs) coprecipitates with SEK1/MKK4 and SAPK/JNK, supporting a role of DNA-PK(cs) in SAPK/JNK activation. In this process, Rho GTPases are involved since inhibition of Rho impairs MMS-induced signaling to SAPK/JNK. The data show that sensing of DNA damage by DNA-PK(cs) and CSB causes a delayed SEK1/MKK4-mediated dual phosphorylation of SAPK/JNK.
Mol Biol Cell 2006 Feb
PMID:Late activation of stress kinases (SAPK/JNK) by genotoxins requires the DNA repair proteins DNA-PKcs and CSB. 1631 74

In this study, we observed the occurrence of TRBV8.1-DB2.1 V(D)J recombination in murine fetal thymus organ culture (FTOC), in which the thymic microenvironment is mimicked. Since ionizing radiation affects T-cell development, we irradiated FTOCs with gamma rays to evaluate the modulation of genes implicated in TRBV8.1-BD2.1 rearrangements. The nylon cDNA microarray method was employed to monitor the expression of 9216 genes, which were organized in coexpression clusters. Clustering analysis showed similar expression profiling of genes implicated in the V(D)J recombination and DNA double strand break (DSB) repair processes such as XRCC4, RAG-2, Artemis and DNA-PK-cs, thus suggesting overlap between the two processes. The RUNX3 gene, whose coded protein binds to the enhancers of TR genes, was also modulated and the DNA cross-linking LR1 gene, which plays a role in the opening of hairpin DNA structures and whose expression pattern is similar to Artemis, may play a role in the control of V(D)J recombination. Furthermore, our data demonstrate that the FTOC model system and cDNA microarray method are useful tools to evidentiate genes that may play a role in both processes V(D)J recombination and DNA repair.
Mol Immunol 2006 Feb
PMID:Hybridization signatures of gamma-irradiated murine fetal thymus organ culture (FTOC) reveal modulation of genes associated with T-cell receptor V(D)J recombination and DNA repair. 1633 89

RAG1 and RAG2 cleave DNA to generate blunt signal ends and hairpin coding ends at antigen receptor loci in lymphoid cells. During V(D)J recombination, repair of these RAG-generated double-strand breaks (DSBs) by the nonhomologous end-joining (NHEJ) pathway contributes substantially to the antigen receptor diversity necessary for immune system function, although recent evidence also supports the ability of RAG-generated breaks to undergo homology-directed repair (HDR). We have determined that RAG-generated chromosomal breaks can be repaired by pathways other than NHEJ in mouse embryonic stem (ES) cells, although repair by these pathways occurs at a significantly lower frequency than NHEJ. HDR frequency was estimated to be >or=40-fold lower than NHEJ frequency for both coding end and signal end reporters. Repair by single-strand annealing was estimated to occur at a comparable or lower frequency than HDR. As expected, V(D)J recombination was substantially impaired in cells deficient for the NHEJ components Ku70, XRCC4, and DNA-PKcs. Concomitant with decreased NHEJ, RAG-induced HDR was increased in each of the mutants, including cells lacking DNA-PKcs, which has been implicated in hairpin opening. HDR was increased to the largest extent in Ku70-/- cells, implicating the Ku70/80 DNA end-binding protein in regulating pathway choice. Thus, RAG-generated DSBs are typically repaired by the NHEJ pathway in ES cells, but in the absence of NHEJ components, a substantial fraction of breaks can be efficiently channeled into alternative pathways in these cells.
Mol Cell Biol 2006 Jan
PMID:Alternative pathways for the repair of RAG-induced DNA breaks. 1635 85

Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genomic integrity. In mammalian cells, DSBs are preferentially repaired by the non-homologous end-joining pathway relying on DNA-PK activity, but other mechanisms may promote end-joining. We previously described a DSB repair pathway that requires synapsis of DNA ends by poly(ADP-ribose) polymerase-1 (PARP-1) and ligation by the XRCC1/DNA ligase III complex (XL). Here, the repair of non-ligatable DNA ends by this pathway was examined in human cell extracts. The phosphorylation of the 5'-terminal end was shown to represent a limiting step for the repair process. Polynucleotide kinase (hPNK) was identified as the 5'-DNA kinase associated with the PARP-1-dependent end-joining pathway because (i) hPNK was co-recruited to DNA ends together with PARP-1 and XL, (ii) ligation of 5'-OH terminal breaks was compromised in hPNK-depleted extracts and restored upon addition of recombinant hPNK, and (iii) recombinant hPNK was necessary for end-joining of 5'-OH terminal breaks reconstituted with the PARP-1/XL complex. Also, using an assay enabling us to follow the ligation kinetics of each strand of a DSB, we established that the two strands at the junction can be processed and joined independently, so that one strand can be ligated without a ligatable nick on the other strand at the DSB site. Taken together these results reveal functional parallels between the PARP-1 and DNA-PK-dependent end-joining processes.
J Mol Biol 2006 Feb 17
PMID:Involvement of polynucleotide kinase in a poly(ADP-ribose) polymerase-1-dependent DNA double-strand breaks rejoining pathway. 1636 63

Recent studies suggest the possibility of a direct antiangiogenic effect of anti-epidermal growth factor receptor (EGFR) drugs due to the presence of EGFR on endothelial cells. The aim of this study was to analyze the direct effect on endothelial cells of associating EGFR targeting, vascular endothelial growth factor receptor (VEGFR)-2 targeting, and irradiation. We examined both the cytotoxic effects and the effect on molecular markers resulting from the combined action of gefitinib (Iressa; anti-EGFR), ZM317450 [VEGFR tyrosine kinase inhibitor (VTKI); anti-VEGFR-2], and irradiation (radiation therapy) on HMME7 cells, an immortalized microvascular endothelial cell of human origin. The presence of a functional EGFR pathway sensitive to gefitinib was shown in HMME7 cells (gefitinib-induced decrease in phospho-EGFR, phospho-p42/p44, and phospho-Akt). The stimulation of VEGFR-2 pathway led to an increase in Akt phosphorylation that was inhibited by VTKI. Of note, a post-radiation therapy induction of phospho-p42/p44 was observed on HMME7 cells, and this effect was inhibited by a pretreatment with gefitinib. Based on combination indexes (Chou and Talalay analyses), the associations gefitinib-radiation therapy, VTKI-radiation therapy, VTKI-gefitinib, and gefitinib-VTKI-radiation therapy were found to be additive, slightly synergistic, and markedly synergistic, respectively, for the cytotoxicity on HMME7 cells. Among molecular explanatory factors that were examined, the combination gefitinib-radiation therapy totally abolishes DNA-dependent protein kinase expression, and gefitinib attenuates the radiation therapy-induced enhancement of ERCC1 and augments the VTKI-induced CD95 enhancement. The existence of a radiation therapy-dependent neoangiogenesis may be related to the induction of EGFR pathway in endothelial cells, a phenomenon that can be attenuated by anti-EGFR drugs like gefitinib. In complement to the direct antitumor effects of radiation therapy and anti-EGFR drugs, a strong antiangiogenic effect may be obtained with therapeutic strategies combining radiation therapy with EGFR and VEGFR-2 targeting agents.
Mol Cancer Ther 2005 Dec
PMID:Response of endothelial cells to a dual tyrosine kinase receptor inhibition combined with irradiation. 1637 11

Here we determined which radiation-responsive genes were altered in radioresistant CEM/IR and FM3A/IR variants, which showed higher resistance to irradiation than parental human leukemia CEM and mouse mammary carcinoma FM3A cells, respectively and studied if radioresistance observed after radiotherapy could be restored by inhibition of protein kinase A. The expressions of DNA-PKcs, Ku70/80, Rad51 and Rad54 genes that related to DNA damage repair, and Bcl-2 and NF-kappaB genes that related to antiapoptosis, were up-regulated, but the expression of proapototic Bax gene was down-regulated in the radioresistant cells as compared to each parental counterpart. We also revealed that the combined treatment of radiation and the inhibitor of protein kinase A (PKA) to these radioresistant cells resulted in synergistic inhibition of DNA-PK, Rad51 and Bcl-2 expressions of the cells, and consequently restored radiosensitivity of the cells. Our results propose that combined treatment with radiotherapy and PKA inhibitor can be a novel therapeutic strategy to radioresistant cancers.
Exp Mol Med 2005 Dec 31
PMID:Radiosensitization by targeting radioresistance-related genes with protein kinase A inhibitor in radioresistant cancer cells. 1639 22

Genome stability in eukaryotic cells is maintained through efficient DNA damage repair pathways, which have to access and utilize chromatin as their natural template. Here we investigate the role of chromatin assembly factor 1 (CAF-1) and its interacting protein, PCNA, in the response of quiescent human cells to DNA double-strand breaks (DSBs). The expression of CAF-1 and PCNA is dramatically induced in quiescent cells upon the generation of DSBs by the radiomimetic drug bleocin (a bleomycin compound) or by ionizing radiation. This induction depends on DNA-PK. CAF-1 and PCNA are recruited to damaged chromatin undergoing DNA repair of single- and double-strand DNA breaks by the base excision repair and nonhomologous end-joining pathways, respectively, in the absence of extensive DNA synthesis. CAF-1 prepared from repair-proficient quiescent cells after induction by bleocin mediates nucleosome assembly in vitro. Depletion of CAF-1 by RNA interference in bleocin-treated quiescent cells in vivo results in a significant loss of cell viability and an accumulation of DSBs. These results support a novel and essential role for CAF-1 in the response of quiescent human cells to DSBs, possibly by reassembling chromatin following repair of DNA strand breaks.
Mol Cell Biol 2006 Mar
PMID:Induction of CAF-1 expression in response to DNA strand breaks in quiescent human cells. 1647 3

The epidermal growth factor receptor (EGFR) is an important target for cancer therapy. We previously showed that the EGFR inhibitor gefitinib modulated repair of DNA damage following exposure to cisplatin and etoposide involving the DNA-dependent protein kinase (DNA-PK) pathway. In this study, we specifically investigated the effect of EGFR inhibition by gefitinib on functional activity of DNA-PK in cancer cell lines and the interaction between EGFR and DNA-PK. The effects of DNA-PK inhibition by wortmannin and small interfering RNA to the catalytic subunit of DNA-PK (DNA-PK(CS)) on cell proliferation and DNA interstrand cross-link repair were investigated in the human MCF-7 breast cancer cell line and compared with the effects of gefitinib. DNA-PK activity was quantitated and expression measured by immunoblotting following gefitinib treatment. Immunoprecipitation experiments were done with and without gefitinib in MCF-7 cells, the AR42J pancreas cell line with high EGFR, and the human MDA-453 breast cancer cell line expressing low EGFR. Nuclear and cytoplasmic extracts were immunoblotted with antibody to DNA-PK(CS) to determine if gefitinib treatment altered cellular expression. Reduction of DNA-PK activity by wortmannin and expression by small interfering RNA to DNA-PK(CS) sensitized cells to cisplatin and inhibited repair of cisplatin-induced interstrand cross-links. Gefitinib treatment reduced DNA-PK activity in MCF-7 and AR42J but not MDA-453 cells. Immunoprecipitation experiments showed interaction between EGFR and DNA-PK(CS) in a dose-dependent and time-dependent manner following gefitinib treatment in MCF-7 and AR42J but not MDA-453 cells. Gefitinib treatment reduced nuclear expression and increased cytosolic expression of DNA-PK(CS) in MCF-7 and AR42J but not MDA-453 cells. Treatment with gefitinib modulates association of EGFR and DNA-PK(CS). This is correlated with decreased function of DNA-PK(CS). Inhibition of DNA-PK(CS) may be an important factor in sensitization to chemotherapy and radiation following treatment with inhibitors of the EGFR pathway.
Mol Cancer Ther 2006 Feb
PMID:Interaction of the epidermal growth factor receptor and the DNA-dependent protein kinase pathway following gefitinib treatment. 1650 93


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