Rejoining of DNA double-strand breaks in X-irradiated CHO cells studied by constant- and graded-field gel electrophoresis

Abstract

Induction and repair of double-strand breaks (dsb) were measured in exponentially growing CHO-10A cells using the constant- and graded-field gel electrophoresis. Dsb repair was studied after an X-ray dose of 60 Gy. The repair curve obtained was biphasic with the respective halftimes of tau1 = 3.8 +/- 0.9 and tau2 = 118 +/- 30 min. The number of non-reparable dsb was measured for X-ray doses up to 180Gy and was found to be only a small fraction (14%) of all nonrejoinable breaks determined previously using the alkaline unwinding technique. The ratio of non-reparable dsb to the number of lethal events calculated from survival curves is 0.14 :1. This result indicates that for CHO cells non-reparable dsb represent only a small fraction of lethal damage. This is in line with the cytogenetic observation that cell killing mainly results from mis-rejoined events (i.e. exchange aberrations, translocations, interstitial deletions). The kinetics of dsb rejoining were found to be independent of the size of the fragments involved (between 1 and 10 Mbp). In addition, the rejoining kinetics of DNA fragments 1Mbp did not show the formation of new DNA fragments with time after irradiation indicating the absence of programmed cell death in irradiated CHO cells.