An assay system was developed to detect changes of restriction fragment profiles obtained after pulsed-field gel electrophoresis (PFGE) for DNA from mammalian cells that were irradiated and incubated for repair. DNA was prepared from irradiated log-phase human melanoma cells (MRT 221) after incubation for repair (6 h) and was digested with the rare-cutting restriction enzyme NotI (RE) prior to PFGE separation. DNA-fragment size distributions were compared to the respective PFGE profiles from unirradiated controls. After doses of 5 and 10 Gy (plus a 6-h incubation for repair), the relative amount of DNA retained in the plug during PFGE was increased. For higher doses (30 and 60 Gy), this phenomenon was superimposed by the residual fragmentation (25-30% of the initial breakage of 0.42 dsb/100Mbp/Gy). Since irradiated cells accumulated in S phase during incubation for repair, a correction for the reduced electrophoretic migration of DNA from S-phase cells was necessary, and a 0.5% increase in DNA retention per 1% S-phase increment was found. However, the % retention for the 5 Gy plus repair sample was significantly higher than for the S-phase adjusted control (p < 0.01). Radiation induced DNA-protein crosslinks cannot account for the observed phenomenon because of the extensive proteolysis in DNA preparation, and also a loss of restriction enzyme recognition sites appear to be an unlikely explanation from simple quantitative considerations. Based on the recent observation of misrejoining during dsb repair, it is proposed that the incorrect joining of DNA ends also causes a more random distribution of replicating DNA in restriction fragments derived from cells in S phase after incubation for repair. This process would necessarily increase the proportion of DNA unable to migrate in PFGE.

Migration patterns in pulsed-field electrophoresis of DNA restriction fragments from log-phase mammalian cells after irradiation and incubation for repair / Latz, D; Dewey, Wc; Flentje, M; Lohr, F; Wenz, F; Weber, Kj. - In: INTERNATIONAL JOURNAL OF RADIATION BIOLOGY. - ISSN 0955-3002. - 70:6(1996), pp. 637-646.

Migration patterns in pulsed-field electrophoresis of DNA restriction fragments from log-phase mammalian cells after irradiation and incubation for repair

Lohr F;
1996

Abstract

An assay system was developed to detect changes of restriction fragment profiles obtained after pulsed-field gel electrophoresis (PFGE) for DNA from mammalian cells that were irradiated and incubated for repair. DNA was prepared from irradiated log-phase human melanoma cells (MRT 221) after incubation for repair (6 h) and was digested with the rare-cutting restriction enzyme NotI (RE) prior to PFGE separation. DNA-fragment size distributions were compared to the respective PFGE profiles from unirradiated controls. After doses of 5 and 10 Gy (plus a 6-h incubation for repair), the relative amount of DNA retained in the plug during PFGE was increased. For higher doses (30 and 60 Gy), this phenomenon was superimposed by the residual fragmentation (25-30% of the initial breakage of 0.42 dsb/100Mbp/Gy). Since irradiated cells accumulated in S phase during incubation for repair, a correction for the reduced electrophoretic migration of DNA from S-phase cells was necessary, and a 0.5% increase in DNA retention per 1% S-phase increment was found. However, the % retention for the 5 Gy plus repair sample was significantly higher than for the S-phase adjusted control (p < 0.01). Radiation induced DNA-protein crosslinks cannot account for the observed phenomenon because of the extensive proteolysis in DNA preparation, and also a loss of restriction enzyme recognition sites appear to be an unlikely explanation from simple quantitative considerations. Based on the recent observation of misrejoining during dsb repair, it is proposed that the incorrect joining of DNA ends also causes a more random distribution of replicating DNA in restriction fragments derived from cells in S phase after incubation for repair. This process would necessarily increase the proportion of DNA unable to migrate in PFGE.
70
6
637
646
Migration patterns in pulsed-field electrophoresis of DNA restriction fragments from log-phase mammalian cells after irradiation and incubation for repair / Latz, D; Dewey, Wc; Flentje, M; Lohr, F; Wenz, F; Weber, Kj. - In: INTERNATIONAL JOURNAL OF RADIATION BIOLOGY. - ISSN 0955-3002. - 70:6(1996), pp. 637-646.
Latz, D; Dewey, Wc; Flentje, M; Lohr, F; Wenz, F; Weber, Kj
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1172423
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