Molecular Analysis of Base Damage Clustering Associated with a Site-Specific Radiation-Induced DNA Double-Strand Break

Department of Nuclear Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.
Radiation Research (Impact Factor: 2.91). 12/2006; 166(5):767-81. DOI: 10.1667/RR0628.1
Source: PubMed


Base damage flanking a radiation-induced DNA double-strand break (DSB) may contribute to DSB complexity and affect break repair. However, to date, an isolated radiation-induced DSB has not been assessed for such structures at the molecular level. In this study, an authentic site-specific radiation-induced DSB was produced in plasmid DNA by triplex forming oligonucleotide-targeted (125)I decay. A restriction fragment terminated by the DSB was isolated and probed for base damage with the E. coli DNA repair enzymes endonuclease III and formamidopyrimidine-DNA glycosylase. Our results demonstrate base damage clustering within 8 bases of the (125)I-targeted base in the DNA duplex. An increased yield of base damage (purine > pyrimidine) was observed for DSBs formed by irradiation in the absence of DMSO. An internal control fragment 1354 bp upstream from the targeted base was insensitive to enzymatic probing, indicating that the damage detected proximal to the DSB was produced by the (125)I decay that formed the DSB. Gas chromatography-mass spectrometry identified three types of damaged bases in the approximately 32-bp region proximal to the DSB. These base lesions were 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine. Finally, evidence is presented for base damage >24 bp upstream from the (125)I-decay site that may form via a charge migration mechanism.

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    • "To explain these differences, it is tempting to speculate that the damage produced by 365 nm and 405 nm laser light is highly complex, possibly reflecting a high density of lesions induced, and as a consequence the reparability of these complex DNA damage sites is reduced, leading to persistence of XRCC1 at the damage sites. Furthermore it is known that complex damage sites, also known as clustered DNA damage sites which contain two or more lesions within one or two turns of the DNA, are difficult to repair [60–63]. The differences seen only highlight the need for essential details of the laser irradiation settings to be given for better comparison of the findings. "
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    ABSTRACT: The formation of DNA lesions poses a constant threat to cellular stability. Repair of endogenously and exogenously produced lesions has therefore been extensively studied, although the spatiotemporal dynamics of the repair processes has yet to be fully understood. One of the most recent advances to study the kinetics of DNA repair has been the development of laser microbeams to induce and visualize recruitment and loss of repair proteins to base damage in live mammalian cells. However, a number of studies have produced contradictory results that are likely caused by the different laser systems used reflecting in part the wavelength dependence of the damage induced. Additionally, the repair kinetics of laser microbeam induced DNA lesions have generally lacked consideration of the structural and chemical complexity of the DNA damage sites, which are known to greatly influence their reparability. In this review, we highlight the key considerations when embarking on laser microbeam experiments and interpreting the real time data from laser microbeam irradiations. We compare the repair kinetics from live cell imaging with biochemical and direct quantitative cellular measurements for DNA repair.
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    • "While the precise chemical complexity of the different DSB ends was not clearly defined, it was postulated that simple DSBs should be easier to repair than DSBs with more complex structures, for instance when several lesions are proximal to the DSB ends. Insights into the structure and chemical complexity of DSBs (12–15) were first revealed from analysis of the chemical composition of radioactive-iodine-induced DSB ends, which are complex (14). Many of these DSBs possess not only single-stranded overhangs of variable length but also a high frequency of oxidized base modifications and abasic sites directly upstream of the DSB ends. "
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