Abstract
BackgroundDNA double strand breaks induced by DNA damaging agents, such ionizing radiation, are repaired by multiple DNA repair pathways including non-homologous end-joining (NHEJ) repair and homologous recombination (HR) repair. ATM-dependent DNA damage checkpoint regulates a part of DNA repair pathways, however, the exact role of ATM activity remains to be elucidated. In order to define the molecular structure of DNA double strand breaks requiring ATM activity we examined repair of DNA double strand breaks induced by different restriction endonucleases in normal human diploid cells treated with or without ATM-specific inhibitor.ResultsSynchronized G1 cells were treated with various restriction endonucleases. DNA double strand breaks were detected by the foci of phosphorylated ATM at serine 1981 and 53BP1. DNA damage was detectable 2 hours after the treatment, and the number of foci decreased thereafter. Repair of the 3'-protruding ends created by Pst I and Sph I was efficient irrespective of ATM function, whereas the repair of a part of the blunt ends caused by Pvu II and Rsa I, and 5'-protruding ends created by Eco RI and Bam HI, respectively, were compromised by ATM inhibition.ConclusionsOur results indicate that ATM-dependent pathway plays a pivotal role in the repair of a subset of DNA double strand breaks with specific end structures.
Highlights
Introduction of restriction endonucleases by electroporationSynchronized cells were washed with phosphate-buffered saline (PBS) twice, collected by trypsinization and resuspended in PBS at a concentration of 2 × 106/ml
DNA double strand breaks are repaired by two major DNA repair pathways, which are non-homologous end-joining (NHEJ) and homologous recombination (HR) [1,2,3,4,5,6]
We introduced various restriction endonucleases into G1-synchronized normal human fibroblast-like cells, and DNA double strand breaks were successfully detected by phosphorylated ATM foci and 53 BP1 foci
Summary
Introduction of restriction endonucleases by electroporationSynchronized cells were washed with phosphate-buffered saline (PBS) twice, collected by trypsinization and resuspended in PBS at a concentration of 2 × 106/ml. Immunofluorescence Cells cultured on coverslips were fixed with 4% formaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 5 min, and were washed extensively with phosphatebuffered saline (PBS). DNA double strand breaks induced by DNA damaging agents, such ionizing radiation, are repaired by multiple DNA repair pathways including non-homologous end-joining (NHEJ) repair and homologous recombination (HR) repair. In order to define the molecular structure of DNA double strand breaks requiring ATM activity we examined repair of DNA double strand breaks induced by different restriction endonucleases in normal human diploid cells treated with or without ATM-specific inhibitor. Ionizing radiation induces various types of DNA damage, among which DNA double strand breaks show the most detrimental effects on living cells.
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