DNA Double-Strand Break Damage and Repair Assessed by Pulsed-Field Gel Electrophoresis

Joshi, Neha S.; Grant, Stephen G.

doi:10.1385/1-59259-840-4:121

Cited by 9 publications

(17 citation statements)

References 21 publications

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“…Accordingly, we have shown that MMS or bleomycin treatments of sea urchin embryos induce cell cycle delay or arrest as evidenced by the delay or absence of cytokinesis correlated with delay or absence of the peak of CDK1/cyclin B activation and CDK1 tyrosine dephosphorylation. The DNA from treated embryos presents the characteristic lesions described generally for MMS or bleomycin [28,29]. The embryos are also able to activate the repair of damage, as demonstrated by pulsed-field analysis of the DNA state.…”

Section: Discussionmentioning

confidence: 94%

“…Analysis of DNA damage and repair in vivo. The DNA profile was analyzed by pulse-field gel electrophoresis (PFGE) [28]. At 20 min after fertilization, the embryo suspension was treated for 30 min in the presence of different genotoxic agents at the indicated concentrations.…”

Section: Introductionmentioning

confidence: 99%

“…Gels were run at 148C with increasing pulse times from 60 to 240 s over 24 h at 100 V. DNA was revealed by ethidium bromide staining and quantified by densitometry. Regression analysis of DNA fragmentation was realized and expressed as the fraction of activity released (FAR), which is the ratio of fragmented DNA to total DNA [28]. DNA repair activity in vitro.…”

Section: Introductionmentioning

confidence: 99%

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Sea urchin embryo as a model for analysis of the signaling pathways linking DNA damage checkpoint, DNA repair and apoptosis

Bouffant

Cormier

Cueff

et al. 2007

Cell. Mol. Life Sci.

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DNA integrity checkpoint control was studied in the sea urchin early embryo. Treatment of the embryos with genotoxic agents such as methyl methanesulfonate (MMS) or bleomycin induced the activation of a cell cycle checkpoint as evidenced by the occurrence of a delay or an arrest in the division of the embryos and an inhibition of CDK1/cyclin B activating dephosphorylation. The genotoxic treatment was shown to induce DNA damage that depended on the genotoxic concentration and was correlated with the observed cell cycle delay. At low genotoxic concentrations, embryos were able to repair the DNA damage and recover from checkpoint arrest, whereas at high doses they underwent morphological and biochemical changes characteristic of apoptosis. Finally, extracts prepared from embryos were found to be capable of supporting DNA repair in vitro upon incubation with oligonucleotides mimicking damage. Taken together, our results demonstrate that sea urchin early embryos contain fully functional and activatable DNA damage checkpoints. Sea urchin embryos are discussed as a promising model to study the signaling pathways of cell cycle checkpoint, DNA repair and apoptosis, which upon deregulation play a significant role in the origin of cancer.

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Section: Discussionmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Sea urchin embryo as a model for analysis of the signaling pathways linking DNA damage checkpoint, DNA repair and apoptosis

Bouffant

Cormier

Cueff

et al. 2007

Cell. Mol. Life Sci.

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“…In PFGE, intact human chromosomes are not able to migrate into the gel. However, when chromosomes are fragmented as a result of DNA damage, shorter pieces of chromosomes can enter the gel (Blocher et al, 1989;Joshi and Grant, 2005). Control siRNA-treated cells did not show an increase in fragmented DNA in response to HU (Fig.…”

Section: Timeless-tipin Is Involved In Replication Fork Stabilizationmentioning

confidence: 94%

“…Preparation and pulsed-field gel electrophoresis (PFGE) of chromosomal DNA were performed as described (Blocher et al, 1989;Joshi and Grant, 2005), using CHEF-DR II system (Bio-Rad) at the following settings. Block 1: field strength, 1.9 V/cm; initial and final switch times, 30 and 120 seconds, respectively; running temperature, 14°C; pump speed, 70; running time, 30 hours.…”

Section: Pulsed-field Gel Electrophoresismentioning

confidence: 99%

Human Timeless and Tipin stabilize replication forks and facilitate sister-chromatid cohesion

Leman

Noguchi

Lee

et al. 2010

Journal of Cell Science

116

161

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SummaryThe Timeless-Tipin protein complex has been reported to be important for replication checkpoint and normal DNA replication processes. However, the precise mechanisms by which Timeless-Tipin preserves genomic integrity are largely unclear. Here, we describe the roles of Timeless-Tipin in replication fork stabilization and sister chromatid cohesion. We show in human cells that Timeless is recruited to replication origin regions and dissociate from them as replication proceeds. Cdc45, which is known to be required for replication fork progression, shows similar patterns of origin association to those of Timeless. Depletion of Timeless-Tipin causes chromosome fragmentation and defects in damage repair in response to fork collapse, suggesting that it is required for replication fork maintenance under stress. We also demonstrate that depletion of Timeless-Tipin impairs sister chromatid cohesion and causes a defect in mitotic progression. Consistently, Timeless-Tipin co-purifies with cohesin subunits and is required for their stable association with chromatin during S phase. Timeless associates with the cohesion-promoting DNA helicase ChlR1, which, when overexpressed, partially alleviates the cohesion defect of cells depleted of Timeless-Tipin. These results suggest that Timeless-Tipin functions as a replication fork stabilizer that couples DNA replication with sister chromatid cohesion established at replication forks.

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The influence of AKT isoforms on radiation sensitivity and DNA repair in colon cancer cell lines

et al. 2013

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In response to ionizing radiation, several signaling cascades in the cell are activated to repair the DNA breaks, prevent apoptosis, and keep the cells proliferating. AKT is important for survival and proliferation and may also be an activating factor for DNA-PKcs and MRE11, which are essential proteins in the DNA repair process. AKT (PKB) is hyperactivated in several cancers and is associated with resistance to radiotherapy and chemotherapy. There are three AKT isoforms (AKT1, AKT2, and AKT3) with different expression patterns and functions in several cancer tumors. The role of AKT isoforms has been investigated in relation to radiation response and their effects on DNA repair proteins (DNA-PKcs and MRE11) in colon cancer cell lines. The knockout of AKT1 and/or AKT2 affected the radiation sensitivity, and a deficiency of both isoforms impaired the rejoining of radiation-induced DNA double strand breaks. Importantly, the active/phosphorylated forms of AKT and DNA-PKcs associate and exposure to ionizing radiation causes an increase in this interaction. Moreover, an increased expression of both DNA-PKcs and MRE11 was observed when AKT expression was ablated, yet only DNA-PKcs expression influenced AKT phosphorylation. Taken together, these results demonstrate a role for both AKT1 and AKT2 in radiotherapy response in colon cancer cells involving DNA repair capacity through the nonhomologous end joining pathway, thus suggesting that AKT in combination with DNA-PKcs inhibition may be used for radiotherapy sensitizing strategies in colon cancer.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-013-1465-9) contains supplementary material, which is available to authorized users.

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DNA Double-Strand Break Damage and Repair Assessed by Pulsed-Field Gel Electrophoresis

Cited by 9 publications

References 21 publications

Sea urchin embryo as a model for analysis of the signaling pathways linking DNA damage checkpoint, DNA repair and apoptosis

Sea urchin embryo as a model for analysis of the signaling pathways linking DNA damage checkpoint, DNA repair and apoptosis

Human Timeless and Tipin stabilize replication forks and facilitate sister-chromatid cohesion

The influence of AKT isoforms on radiation sensitivity and DNA repair in colon cancer cell lines

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