The contribution of thermally labile sugar lesions to DNA double-strand break formation in cells grown in the presence of BrdU

Abstract

Purpose: Radiosensitization by bromodeoxyuridine (BrdU) is commonly attributed to an increase in the yield of double-strand breaks (DSB) in the DNA and an associated decrease in the reparability of these lesions. Radiation chemistry provides a mechanism for the increased yield of DSB through the generation, after bromine loss, of a highly reactive uracilyl radical that attacks the sugar moiety of the nucleotide to produce a single-strand break (SSB). The effects underpinning DSB repair inhibition remain, in contrast, incompletely characterized. A possible source of reduced reparability is a change in the nature or complexity of the DSB in BrdU-substituted DNA. Recent studies show that DSB-complexity or DSB-nature may also be affected by the presence within the cluster of thermally labile sugar lesions (TLSL) that break the DNA backbone only if they chemically evolve to SSB, a process thought to occur within the first hour post-irradiation. Since BrdU radiosensitization might be associated with increased yields and reduced reparability of DSB, we investigated whether BrdU underpins these effects by shifting the balance in the generation of TLSL.

Methods and materials: We employed asymmetric-field-inversion gel electrophoresis (AFIGE), a pulsed-field gel electrophoresis (PFGE) method to quantitate DSB in a battery of five cells lines grown in the presence of different concentrations of BrdU. We measured specifically the yields of promptly forming DSB (prDSB) using low temperature lysis protocols, and the yields of total DSB (tDSB = prDSB + tlDSB; tlDSB form after evolution to SSB of TLSL) using high temperature lysis protocols.

Results: We report that incorporation of BrdU generates similar increases in the formation of tlDSB and prDSB, but variations are noted among the different cell lines tested.

Conclusions: The similar increase in the yields of tlDSB and prDSB in BrdU substituted DNA showed that shifts in the yields of these forms of lesions could not be invoked to explain BrdU radiosensitization.

Keywords::

• DNA double-strand breaks (DSB)
• ionizing radiation (IR)
• BrdU
• halogenated pyrimidines
• thermally labile sugar lesions (TLSL)
• radiation chemistry
• pulsed-field gel electrophoresis (PFGE)

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by grants from the “Bundesministerium für Bildung und Forschungˮ (BMBF: 02NUK005C and 03NUK001B) and by a grant from the “Bundesministerium für Wirtschaft und Technologieˮ (BMWi: ESA-AO-08-IBER, 50WB1229).

Supplementary material available online

Supplementary Figure 1.