Strand Breaks and 5′ End-groups in DNA of Irradiated Thymocytes

Coquerelle, Thérèse; Bopp, Annemarie; Keßler, Bernd; Hagen, U.

doi:10.1080/09553007314551251

Cited by 70 publications

(39 citation statements)

References 17 publications

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“…Deletion of PNK in fission yeast resulted in cellular hypersensitivity to ionizing radiation and camptothecin, a topoisomerase I inhibitor. Both of these agents are capable of generating DNA SSB and DSB with termini requiring either or both PNK activities (12)(13)(14)(15). RNA interference (RNAi) silencing of hPNK in A549 human lung carcinoma cells yielded a similar response to that seen in yeast (16).…”

Section: Introductionmentioning

confidence: 87%

Human Polynucleotide Kinase Participates in Repair of DNA Double-Strand Breaks by Nonhomologous End Joining but not Homologous Recombination

et al. 2007

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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]

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Section: Introductionmentioning

confidence: 87%

Human Polynucleotide Kinase Participates in Repair of DNA Double-Strand Breaks by Nonhomologous End Joining but not Homologous Recombination

et al. 2007

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“…These proteins are sufficient for "simple religation" NHEJ of breaks with compatible termini. Joining of damaged termini is not as well understood, but is important because DNA damaging agents like ionizing radiation and reactive oxygen species typically create DSBs with "dirty" ends [5][6][7][8].…”

Section: Introductionmentioning

confidence: 99%

Evidence that base stacking potential in annealed 3′ overhangs determines polymerase utilization in yeast nonhomologous end joining

Daley

Wilson

2008

DNA Repair

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Nonhomologous end joining (NHEJ) directly rejoins DNA double-strand breaks (DSBs) when recombination is not possible. In Saccharomyces cerevisiae, the DNA polymerase Pol4 is required for gap filling when a short 3' overhang must prime DNA synthesis. Here, we examined further end variations to test specific hypotheses regarding Pol4 usage in NHEJ in vivo. Surprisingly, Pol4 dependence at 3' overhangs was reduced when a nonhomologous 5' flap nucleotide was present across from the gap, even though the mismatched nucleotide was resynthesized, not incorporated. In contrast, a gap with a 5' deoxyribosephosphate (dRP) was as Pol4-dependent as a gap with a 5' phosphate, demonstrating the importance of the downstream base in relaxing the Pol4 requirement. Combined with prior observations of Pol4-independent NHEJ of nicks with 5' hydroxyls, we suggest that base stacking interactions across the broken strands can stabilize a joint, allowing another polymerase to substitute for Pol4. This model predicts that a unique function of Pol4 is to actively stabilize template strands that lack stacking continuity. We also explored whether NHEJ end processing can occur via short and long patch pathways analogous to base excision repair. Results demonstrated that 5' dRPs could be removed in the absence of Pol4 lyase activity. The 5' flap endonuclease Rad27 was not required for repair in this or any situation tested, indicating that still other NHEJ 5' nucleases must exist.

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“…Thymocytes were irradiated with 10 MeV electrons, suspended in Hank's solution and bubbled with oxygen or nitrogen (see Coquerelle, Bopp, Kessler and Hagen 1973) . To incubate the cells after irradiation a vessel with two compartments was used kept at 37°C by a water-jacket .…”

Section: Methodsmentioning

confidence: 99%

“…From the lysed cells DNA was isolated, and the frequency of double-strand breaks (dsb), of actual single-strand breaks (ssb) and alkali-induced strand breaks (alisb) was determined by means of sedimentation in the analytical ultracentrifuge as described by Coquerelle et al (1973) . On the terminals of the ssb and alisb, the frequency of 3'OH end-groups was evaluated by means of the terminal 182 Correspondence deoxyribonucleotide transferase and the frequency of 5'OH and 5'phosphate end-groups by means of the polynucleotide kinase and acid phosphomonoesterase, as described in detail by Lennartz et al (1975) .…”

Section: Methodsmentioning

confidence: 99%

Modification of End-groups in DNA Strand Breaks of Irradiated Thymocytes during Early Repair

Lennartz

Coquerelle

Hagen

1975

International Journal of Radiation Biology and Related Studies

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Strand Breaks and 5′ End-groups in DNA of Irradiated Thymocytes

Cited by 70 publications

References 17 publications

Human Polynucleotide Kinase Participates in Repair of DNA Double-Strand Breaks by Nonhomologous End Joining but not Homologous Recombination

Human Polynucleotide Kinase Participates in Repair of DNA Double-Strand Breaks by Nonhomologous End Joining but not Homologous Recombination

Evidence that base stacking potential in annealed 3′ overhangs determines polymerase utilization in yeast nonhomologous end joining

Modification of End-groups in DNA Strand Breaks of Irradiated Thymocytes during Early Repair

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