Quantifying DNA damage induced by ionizing radiation and hyperthermia using single DNA molecule imaging

Abstract

Ionizing radiation (IR) is a common mode of cancer therapy, where DNA damage is the major reason of cell death. Here, we use an assay based on fluorescence imaging of single damaged DNA molecules isolated from radiated lymphocytes, to quantify IR induced DNA damage. The assay uses a cocktail of DNA-repair enzymes that recognizes and excises DNA lesions and then a polymerase and a ligase incorporate fluorescent nucleotides at the damage sites, resulting in a fluorescent “spot” at each site. The individual fluorescent spots can then be counted along single stretched DNA molecules and the global level of DNA damage can be quantified. Our results demonstrate that inclusion of the human apurinic/apyrimidinic endonuclease 1 (APE1) in the enzyme cocktail increases the sensitivity of the assay for detection of IR induced damage significantly. This optimized assay also allowed detection of a cooperative increase in DNA damage when IR was combined with mild hyperthermia, which is sometimes used as an adjuvant in IR therapy. Finally, we discuss how the method may be used to identify patients that are sensitive to IR and other types of DNA damaging agents.

Click and Cut: a click chemistry approach to developing oxidative DNA damaging agents

“…Later, a dye (YOYO-1) was used to stain the DNA backbone and individual DNA molecules were stretched on silanized glass coverslips, where image analysis determined the extent of DNA damage by quantifying the incorporation of repaired bases on the single DNA molecule level ( cf . Figure 9C ) ( 51 , 63–65 ). PBMCs were exposed to Cu(II)–TC1 and intracellular SSBs were quantified (Figure 9d ).…”

Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication

“…ChIP-seq analysis showed that cells depleted of Pfh1 not only experience replication fork stalling, but also increased DNA damage as detected by γ-H2A, a marker of DNA damage ( 46 ). To determine if G4 stabilization by PhenDC3 causes ssDNA damage in S. pombe , we measured the formation of DNA lesions in PhenDC3-treated MDRΔ cells using a DNA damage detection assay based on imaging of single DNA molecules ( 43 , 44 ). In this DNA damage detection assay, we labeled the ssDNA breaks formed in genomic DNA isolated from PhenDC3-treated and untreated MDRΔ (YJJ21) cells (Figure 5A ).…”

“…The base excision repair (BER) pathway repairs base lesions caused by for instance oxidation, alkylation and deamination. Recently, a BER-based single-molecule imaging technique was used to identify and quantify ionizing radiation-induced single-stranded DNA (ssDNA) lesions ( 43 ). This technique identifies different types of DNA damages caused by DNA damaging agents based on proficiency of a particular enzyme in a BER enzyme mixture ( 43 , 44 ).…”

Modulating the Heat Stress Response to Improve Hyperthermia-Based Anticancer Treatments

“…The ability of heat to interfere with the function of various DNA repair mechanisms (e.g., homology-directed repair, nonhomologous end-joining, base-excision repair) are known to greatly contribute to the efficacy of DNA-damaging therapies, including chemo and radiotherapy [ 115 ]. Heat may further amplify these effects by inducing various types of DNA damage, including nucleotide modifications and even, albeit controversially, DNA double-strand breaks (DSBs) [ 112 , 116 , 117 , 118 ]. In conclusion, mild HT affects various nuclear processes and sensitizes tumor cells to DNA-damaging therapies.…”