Delayed repletion of O6-methylguanine—DNA methyltransferase resulting in failure to protect the human glioblastoma cell line SF767 from temozolomide-induced cytotoxicity

2003 ◽  
Vol 98 (3) ◽  
pp. 591-598 ◽  
Author(s):  
Yuichi Hirose ◽  
Emiko L. Kreklau ◽  
Leonard C. Erickson ◽  
Mitchel S. Berger ◽  
Russell O. Pieper
Keyword(s):  
Cell Death ◽  
Dna Methyltransferase ◽  
Glioma Cells ◽  
Double Strand Breaks ◽  
Single Strand ◽  
Dna Double Strand Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Fine Print

Object. Temozolomide (TMZ)-induced O6-methylguanine (MG) DNA lesions, if not removed by MG—DNA methyltransferase (MGMT), mispair with thymine, trigger rounds of futile mismatch repair (MMR), and in glioma cells lead to prolonged G2—M arrest and ultimately cell death. Depletion of MGMT by O6-benzylguanine (BG) sensitizes tumor cells to TMZ, and this combination is currently used in clinical trials. The use of the TMZ+BG combination in gliomas, however, is complicated by the prolonged TMZ-induced G2—M arrest, which may delay activation of poorly defined cell death pathways and allow for MGMT repletion and reversal of toxicity. Methods. To address these issues, the actions of TMZ were monitored in DNA MMR-proficient SF767 glioma cells depleted of MGMT by BG, and in cells in which BG was removed at various times after TMZ exposure. In MGMT-depleted cells, TMZ exposure led to DNA single-strand breaks and phosphorylation of cdc2, followed by G2—M arrest, induction of p53/p21, and DNA double-strand breaks. Although DNA single-strand breaks, phosphorylation of cdc2, and G2—M arrest could be reversed by repletion of MGMT up to 5 days after TMZ exposure, TMZ-induced cytotoxicity could only be prevented if MGMT was replenished within 24 hours of the onset of G2—M arrest, and before the creation of DNA double-strand breaks. Conclusions. These results indicate that although SF767 glioma cells undergo a prolonged G2—M arrest in response to TMZ, their ability to escape TMZ-induced cytotoxicity by MGMT repletion is limited to an approximately 24-hour period after the onset of G2—M arrest.

Change in DNA after Exposure to Hydrogen Atoms.

1969 ◽  
Vol 24 (12) ◽  
pp. 1565-1573 ◽  
Author(s):  
H. Jung, ◽  
U. Hagen, ◽  
M. Ullrich, ◽  
E. E. Petersen
Keyword(s):  
Hydrogen Bonds ◽  
Double Strand Breaks ◽  
Single Strand ◽  
Dna Double Strand Breaks ◽  
Hydrogen Atoms ◽  
Γ Irradiation ◽  
Base Damage ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Single Breaks

The action of hydrogen atoms — generated in an electrodeless high frequency gas discharge — on calf thymus DNA in aqueous solution was investigated. The loss of priming activity was compared with the appearance of single strand breaks in native and denatured DNA, double strand breaks, denatured zones, base damage and rupture of hydrogen bonds. The primary lesions after exposure to H atoms and gamma radiation, respectively, are single strand breaks and base damage. Double strand breaks originating from accumulation of single breaks, and rupture of hydrogen bonds caused by single breaks and base damage, were identified as secondary lesions. In relation to strand breaks arising from radical attack on the sugar-phosphate backbone of the DNA molecule, base damage is about 12.5 times more frequent after Η-exposure than after γ-irradiation. It is concluded from this observation, that single strand breaks are the predominant critical lesions responsible for the loss of the functional activity of DNA.

scholarly journals Deinococcus radioduransHD-Pnk, a Nucleic Acid End-Healing Enzyme, Abets Resistance to Killing by Ionizing Radiation and Mitomycin C

2018 ◽  
Vol 200 (17) ◽  
Author(s):  
Brad J. Schmier ◽  
Stewart Shuman
Keyword(s):  
Nucleic Acid ◽  
Ionizing Radiation ◽  
Mitomycin C ◽  
Deinococcus Radiodurans ◽  
Single Strand ◽  
Dna Double Strand Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Key Steps

ABSTRACT5′- and 3′-end healing are key steps in nucleic acid break repair in which 5′-OH and 3′-PO4or 2′,3′-cyclic-PO4ends are converted to 5′-PO4and 3′-OH termini suitable for sealing by polynucleotide ligases. Here, we characterizeDeinococcus radioduransHD-Pnk as a bifunctional end-healing enzyme composed of N-terminal HD (histidine-aspartate) phosphoesterase and C-terminal P-loop polynucleotide kinase (Pnk) domains. HD-Pnk phosphorylates 5′-OH DNA in the presence of ATP and magnesium. HD-Pnk has 3′-phosphatase and 2′,3′-cyclic-phosphodiesterase activity in the presence of transition metals, optimally cobalt or copper, and catalyzes copper-dependent hydrolysis ofp-nitrophenylphosphate. HD-Pnk is encoded by the LIG–PARG–HD-Pnk three-gene operon, which includes polynucleotide ligase and poly(ADP-ribose) glycohydrolase genes. We show that whereas HD-Pnk is inessential forDeinococcusgrowth, its absence sensitizes by 80-fold bacteria to killing by 9 kGy of ionizing radiation (IR). HD-Pnk protein is depleted during early stages of post-IR recovery and then replenished at 15 h, after reassembly of the genome from shattered fragments. ΔHD-Pnk mutant cells are competent for genome reassembly, as gauged by pulsed-field gel electrophoresis. Our findings suggest a role for HD-Pnk in repairing residual single-strand gaps or nicks in the reassembled genome. HD-Pnk-Ala mutations that ablate kinase or phosphoesterase activity sensitizeDeinococcusto killing by mitomycin C.IMPORTANCEEnd healing is a process whereby nucleic acid breaks with “dirty” 3′-PO4or 2′,3′-cyclic-PO4and 5′-OH ends are converted to 3′-OH and 5′-PO4termini that are amenable to downstream repair reactions.Deinococcus radioduransis resistant to massive doses of ionizing radiation (IR) that generate hundreds of dirty DNA double-strand breaks and thousands of single-strand breaks. This study highlightsDeinococcusHD-Pnk as a bifunctional 3′- and 5′-end-healing enzyme that helps protect against killing by IR. HD-Pnk appears to act late in the process of post-IR recovery, subsequent to genome reassembly from shattered fragments. HD-Pnk also contributes to resistance to killing by mitomycin C. These findings are significant in that they establish a role for end-healing enzymes in bacterial DNA damage repair.

scholarly journals Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

2017 ◽  
Vol 37 (24) ◽  
Author(s):  
Sucheta Arora ◽  
Rajashree A. Deshpande ◽  
Martin Budd ◽  
Judy Campbell ◽  
America Revere ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

Sign in / Sign up

Export Citation Format

Share Document