Mechanism of cell killing after ionizing radiation by a dominant negative DNA polymerase beta.

Abstract

Several types of DNA lesion are induced after ionizing irradiation (IR) of which double strand breaks (DSBs) are expected to be the most lethal, although single strand breaks (SSBs) and DNA base damages are quantitatively in the majority. Proteins of the base excision repair (BER) pathway repair these numerous lesions. DNA polymerase beta has been identified as a crucial enzyme in BER and SSB repair (SSBR). We showed previously that inhibition of BER/SSBR by expressing a dominant negative DNA polymerase beta (polbetaDN) resulted in radiosensitization. We hypothesized increased kill to result from DSBs arising from unrepaired SSBs and BER intermediates. We find here higher numbers of IR-induced chromosome aberrations in polbetaDN expressing cells, confirming increased DSB formation. These aberrations did not result from changes in DSB induction or repair of the majority of lesions. SSB conversion to DSBs has been shown to occur during replication. We observed an increased induction of chromatid aberrations in polbetaDN expressing cells after IR, suggesting such a replication-dependence of secondary DSB formation. We also observed a pronounced increase of chromosomal deletions, the most likely cause of the increased kill. After H(2)O(2) treatment, polbetaDN expression only resulted in increased chromatid (not chromosome) aberrations. Together with the lack of sensitization to H(2)O(2), these data further suggest that the additional secondarily induced lethal DSBs resulted from repair attempts at complex clustered damage sites, unique to IR. Surprisingly, the polbetaDN induced increase in residual gammaH2AX foci number was unexpectedly low compared with the radiosensitization or induction of aberrations. Our data thus demonstrate the formation of secondary DSBs that are reflected by increased kill but not by residual gammaH2AX foci, indicating an escape from gammaH2AX-mediated DSB repair. In addition, we show that in the polbetaDN expressing cells secondary DSBs arise in a radiation-specific and partly replication-dependent manner.

• Authors: Sari Neijenhuis, Manon Verwijs-Janssen, Ulla Kasten-Pisula, Gaby Rumping, Kerstin Borgmann, Ekkehard Dikomey, Adrian C Begg, Conchita Vens
• Journal: DNA repair (DNA Repair (Amst)) Vol. 8 Issue 3 Pg. 336-46 (Mar 01 2009) ISSN: 1568-7864 [Print] Netherlands
• PMID: 19059500 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• H2AX protein, human
• Histones
• DNA
• DNA Polymerase beta

Topics

• Cell Death (radiation effects)
• Cell Line, Tumor
• Cell Survival (genetics, radiation effects)
• Chromatids (radiation effects)
• DNA (radiation effects)
• DNA Breaks, Double-Stranded (radiation effects)
• DNA Breaks, Single-Stranded (radiation effects)
• DNA Polymerase beta (physiology)
• DNA Repair (radiation effects)
• DNA Replication
• Dose-Response Relationship, Radiation
• Histones
• Humans
• Oxidative Stress (genetics)
• Radiation, Ionizing