Methods for the Quantification of DNA Double-Strand Breaks Determined from the Distribution of DNA Fragment Sizes Measured by Pulsed-Field Gel Electrophoresis

Cedervall, Björn; Wong, Rosemary; Albright, Norman; Dynlacht, Joseph R.; Lambin, P.; Dewey, William C.

doi:10.2307/3578920

Cited by 85 publications

(48 citation statements)

References 23 publications

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“…(Table 2). This value confirmed our previous results El-Awady et al, 2001) and also agreed well with other data (range 8 -15 Â 10 À12 dsb/Gy/Da) based on PFGE, from which a number of dsb can be calculated when it is combined with either 125 I-decay, analysis of fragment size distribution after high doses, or restriction digest (Blöcher and Pohlit, 1982;Iliakis et al, 1991a, b;Lawrence et al, 1993;Cedervall et al, 1994Cedervall et al, , 1995Löbrich et al, 1994a, b;Löbrich, 1999, 2003). Only, Ruiz de Almodovar et al (1994) found a much higher induction frequency of about 66 dsb Â 10 À12 /Gy/Da.…”

Section: Number Of Induced Dsbsupporting

confidence: 92%

“…Pulsed-field gel electrophoresis (PFGE), currently most widely used, has the advantage to resolve large DNA fragments according to the molecular size permitting, in principle, a direct quantification of dsb. However, the accurate analysis of the profile of the continuous fragment distribution is not trivial Kraxenberger et al, 1994;Cedervall et al, 1995). In addition, PFGE could be complicated by paradoxical migration patterns (Carle et al, 1986;Chu, 1991;Löbrich et al, 1993).…”

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confidence: 99%

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Radiosensitivity of human tumour cells is correlated with the induction but not with the repair of DNA double-strand breaks

2003

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Nine human tumour cell lines (four mammary, one bladder, two prostate, one cervical, and one squamous cell carcinoma) were studied as to whether cellular radiosensitivity is related to the number of initial or residual double-strand breaks (dsb). Cellular sensitivity was measured by colony assay and dsb by means of constant-and graded-field gel electrophoresis (CFGE and GFGE, respectively). The nine tumour cell lines showed a broad variation in cellular sensitivity (SF2 0.17 -0.63). The number of initial dsb as measured by GFGE ranged between 14 and 27 dsb/Gy/diploid DNA content. In contrast, normal fibroblasts raised from skin biopsies of seven individuals showed only a marginal variation with 18 -20 dsb/Gy/diploid DNA content. For eight of the nine tumour cell lines, there was a significant correlation between the number of initial dsb and the cellular radiosensitivity. The tumour cells showed a broad variation in the amount of dsb measured 24 h after irradiation by CFGE, which, however, was not correlated with the cellular sensitivity. This residual damage was found to be influenced not only by the actual number of residual dsb, but also by apoptosis and cell cycle progression which had impact on CFGE measurements. Some cell line strains were able to proliferate even after exposure to 150 Gy while others were found to degrade their DNA. Our results suggest that for tumour cells, in contrast to normal cells, the variation in sensitivity is mainly determined by differences in the initial number of dsb induced.

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Section: Number Of Induced Dsbsupporting

confidence: 92%

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confidence: 99%

Radiosensitivity of human tumour cells is correlated with the induction but not with the repair of DNA double-strand breaks

2003

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“…We monitored the formation of DNA breaks using pulsed-field gel electrophoresis (PFGE), where the distribution of randomly broken DNA molecules can be determined by DNA migration from the high-molecular-weight chromosomal DNA band (31,32). Exponentially growing cultures were exposed to M. albus VOCs and then embedded in a low-melting-point agarose gel in order to prevent the indirect breakage of genomic DNA during the subsequent cell lysis.…”

Section: Resultsmentioning

confidence: 99%

Mycofumigation by the Volatile Organic Compound-Producing Fungus Muscodor albus Induces Bacterial Cell Death through DNA Damage

Alpha

Campos

Jacobs‐Wagner

et al. 2015

Appl Environ Microbiol

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bMuscodor albus belongs to a genus of endophytic fungi that inhibit and kill other fungi, bacteria, and insects through production of a complex mixture of volatile organic compounds (VOCs). This process of mycofumigation has found commercial application for control of human and plant pathogens, but the mechanism of the VOC toxicity is unknown. Here, the mode of action of these volatiles was investigated through a series of genetic screens and biochemical assays. A single-gene knockout screen revealed high sensitivity for Escherichia coli lacking enzymes in the pathways of DNA repair, DNA metabolic process, and response to stress when exposed to the VOCs of M. albus. Furthermore, the sensitivity of knockouts involved in the repair of specific DNA alkyl adducts suggests that the VOCs may induce alkylation. Evidence of DNA damage suggests that these adducts lead to breaks during DNA replication or transcription if not properly repaired. Additional cytotoxicity profiling indicated that during VOC exposure, E. coli became filamentous and demonstrated an increase in cellular membrane fluidity. The volatile nature of the toxic compounds produced by M. albus and their broad range of inhibition make this fungus an attractive biological agent. Understanding the antimicrobial effects and the VOC mode of action will inform the utility and safety of potential mycofumigation applications for M. albus. T he endophytic fungusMuscodor albus (CZ-620) inhibits growth of a broad range of pathogenic fungi and bacteria, as well as some nematode and arthropod species (1-6). The inhibition is achieved exclusively through a complex mixture of volatile organic compounds (VOCs) that M. albus secretes into the headspace of the culture. The volatile compounds emitted by M. albus and other closely related organisms in the genus consist of a combination of short-chain alcohols, organic acids, esters, ketones, and several aromatic hydrocarbons as monitored by gas chromatography-mass spectrometry (GC-MS) (6). The compounds range from two to nine carbons and include both straight and branched-chain varieties. The larger aromatic products are predicted to be sesquiterpenes and derivatives of naphthalene and azulene but have not been confirmed by comparison to standards. Although many fungal species have been reported to produce VOCs, none have demonstrated the wide-ranging bioactivity seen with isolates of M. albus (7,8).The biological function of the toxic compound production by M. albus is unknown. M. albus was first identified as an endophyte, an organism that lives within the inner tissues of plants (9). Although endophytic interactions between fungi and host plants are typically asymptomatic for part, or all, of their life cycle, these can also be commensal relationships. There are examples where fungi improve the host plant's growth, fitness, or response to stress response (10-12). In the case of M. albus, its VOC emissions may serve as a defense mechanism for the host plant against insects or potential pathogens, and these same products ha...

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“…Mammalian cells tolerate several grays of radiation, which is sufficient to induce dozens of breaks simultaneously (12,54). The ability of Ku to anchor newly formed ends to an underlying structure could serve an important function in suppressing illegitimate rejoining of mismatched DNA ends.…”

Section: Discussionmentioning

confidence: 99%

Subnuclear Localization of Ku Protein: Functional Association with RNA Polymerase II Elongation Sites

Dynan

2002

Molecular and Cellular Biology

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Ku is an abundant nuclear protein with an essential function in the repair of DNA double-strand breaks. Various observations suggest that Ku also interacts with the cellular transcription machinery, although the mechanism and significance of this interaction are not well understood. In the present study, we investigated the subnuclear distribution of Ku in normally growing human cells by using confocal microscopy, chromatin immunoprecipitation, and protein immunoprecipitation. All three approaches indicated association of Ku with RNA polymerase II (RNAP II) elongation sites. This association occurred independently of the DNA-dependent protein kinase catalytic subunit and was highly selective. There was no detectable association with the initiating isoform of RNAP II or with the general transcription initiation factors. In vitro protein-protein interaction assays demonstrated that the association of Ku with elongation proteins is mediated, in part, by a discrete C-terminal domain in the Ku80 subunit. Functional disruption of this interaction with a dominantnegative mutant inhibited transcription in vitro and in vivo and suppressed cell growth. These results suggest that association of Ku with transcription sites is important for maintenance of global transcription levels. Tethering of double-strand break repair proteins to defined subnuclear structures may also be advantageous in maintenance of genome stability.The Ku protein is part of the DNA-dependent protein kinase (DNA-PK), which is required for the nonhomologous end-joining pathway of DNA double-strand break (DSB) repair (53, 59). When a break occurs, Ku binds avidly to the DNA ends, translocates inward, and recruits the 470-kDa DNA-PK catalytic subunit (DNA-PKcs) to form an active protein kinase complex (19,26,74). Proteins in this complex cooperate with XRCC4, DNA ligase IV, and other factors to carry out DSB repair (reviewed in reference 22). Ku-deficient cells are sensitive to ionizing radiation and are unable to complete the process of V(D)J recombination, which involves a DSB intermediate.In addition to its well-documented role in DSB repair, Ku appears to have some interaction with the RNA polymerase II (RNAP II) transcription apparatus. Several reports describe the binding of Ku to promoter regions or its ability to regulate transcription of individual genes (reviewed in reference 21 and also see references 11, 25, 31, and 72). Other reports describe interaction of Ku with the general transcription machinery. DNA-PK efficiently phosphorylates RNAP II in vitro (50), and a fraction of Ku resides in RNAP II-containing complexes (41). The importance of these interactions is suggested by the finding that nuclear extracts of Ku-deficient cells exhibit a characteristic transcription defect (70). Transcription of several different promoters is decreased two-to fivefold relative to that of extracts from a matched Ku-containing cell line (70). The defect is entirely at the level of reinitiation and is not seen in assays in which transcription is limited to a ...

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Methods for the Quantification of DNA Double-Strand Breaks Determined from the Distribution of DNA Fragment Sizes Measured by Pulsed-Field Gel Electrophoresis

Cited by 85 publications

References 23 publications

Radiosensitivity of human tumour cells is correlated with the induction but not with the repair of DNA double-strand breaks

Radiosensitivity of human tumour cells is correlated with the induction but not with the repair of DNA double-strand breaks

Mycofumigation by the Volatile Organic Compound-Producing Fungus Muscodor albus Induces Bacterial Cell Death through DNA Damage

Subnuclear Localization of Ku Protein: Functional Association with RNA Polymerase II Elongation Sites

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