Enzymatic probing of chromatin conformation using the comet assay
Creators: name: Thompson, Jennifer Jane
Publisher: name: University of British Columbia
Year: 2009

description: Chromatin structure is thought to play an important role in the response of mammalian cells to ionizing radiation. Structural differences amongst cell types can determine retention of topology after radiation damage, recovery rates of global conformation, repair enzyme accessibility as well as impose limitations on detection of lesions. Chromatin structure may also influence the distribution of radiation-induced DNA lesions within cells. Detection of structural features of cell types that influence response to radiation would be of practical importance in designing predictive assays for tumour response to treatment. In conjunction with the comet assay, a rapid method of DNA damage detection in individual cells, the effects of radiation could be more accurately predicted by assessing not only the quantity of lesions produced but how individual cells 'cope' with X-ray insult, particularly double-strand breaks which cause lethal chromosomal aberrations. To examine the influence of variations in chromatin conformation on response to DNA double-strand breakage, a restriction endonuclease method was developed and optimized as an adjunct to comet analysis. EcoRl cleaves double-stranded DNA at defined sites within the genome. This enzyme was chosen to create DSBs for two cell lines which varied widely in radiosensitivity: Chinese hamster V79 lung fibroblasts and human B lymphoblastoid TK6 cells. Chromatin organization was manipulated by increasing NaCl concentration from 0 to 3 M to progressively remove different histone and non-histone proteins. This approach provided a method to assess the possible importance of accessibility of DNA recognition sites to the enzyme. For these cell lines, no significant differences were observed, suggesting that accessibility to this restriction enzyme was not affected by differences in chromatin "packaging" that have been observed for these cells using other methods. However this general technique provides a potentially useful method for further restriction enzyme or DNA repair enzyme studies. As a related study, DNA repair enzymes for detection of base damage produced by ionizing radiation were examined for their ability to detect additional DNA damage in irradiated cells prepared as comets. Efforts were first made to optimize lysis and enzyme treatment conditions for V79 cells. For this project, alkali lysis and electrophoresis were necessary to detect single-strand breaks created when an endonuclease cleaves the phosphodiester backbone at sites of base damage. A crude extract prepared from the bacterium Micrococcal leuteus was examined for its ability to recognize ionizing radiation-induced base damage and to produce additional SSBs in the comet assay. While additional damage was observed, there was also an increase in background damage in unirradiated samples. Since the crude extract may contain endogenous nucleases that might mask the effects of base damage detection enzymes, a purified sample of Endonuclease III was then obtained and analyzed using the same method. However, in spite of considerable effort, no additional DNA damage was detected in irradiated cells exposed to Endo in, suggesting that unknown factors may be preventing access of the enzyme to base damage sites or that conditions chosen for this study inadvertently affected enzyme activity.