Tests of the Single-hit DNA Damage Model

Spangler, Rudolph; Goddard, Noël L.; Spangler, Douglas N.; Thaler, David S.

doi:10.1016/j.jmb.2009.07.012

Cited by 9 publications

(6 citation statements)

References 49 publications

(60 reference statements)

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“…X-ray irradiation is known to generate mainly strand breaks (single and double stranded), but that could be a rather infrequent event which only matters in a large genome such as the nuclear [20]. This suggestion is in agreement with experiments from Spangler and co-authors [21] who found that DNA damage from γ-irradiation was most consistent with a single-hit mechanism and did not differ in DNA fragments of different size; again suggesting it to be a rather rare event. DNA repair in mtDNA after high doses of irradiation has not been investigated very intensely, most studies instead focused on mutation rates, changes in mtDNA copy numbers [22,23] or found a relaxation in supercoiling of the double stranded mitochondrial DNA [24].…”

Section: Discussionsupporting

confidence: 88%

Telomerase Does Not Improve DNA Repair in Mitochondria upon Stress but Increases MnSOD Protein under Serum-Free Conditions

Martens

Schmid

Akintola

et al. 2019

IJMS

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Telomerase is best known for its function in maintaining telomeres but has also multiple additional, non-canonical functions. One of these functions is the decrease of oxidative stress and DNA damage due to localisation of the telomerase protein TERT into mitochondria under oxidative stress. However, the exact molecular mechanisms behind these protective effects are still not well understood. We had shown previously that overexpression of human telomerase reverse transcriptase (hTERT) in human fibroblasts results in a decrease of mitochondrial DNA (mtDNA) damage after oxidative stress. MtDNA damage caused by oxidative stress is removed via the base excision repair (BER) pathway. Therefore we aimed to analyse whether telomerase is able to improve this pathway. We applied different types of DNA damaging agents such as irradiation, arsenite treatment (NaAsO2) and treatment with hydrogen peroxide (H2O2). Using a PCR-based assay to evaluate mtDNA damage, we demonstrate that overexpression of hTERT in MRC-5 fibroblasts protects mtDNA from H2O2 and NaAsO2 induced damage, compared with their isogenic telomerase-negative counterparts. However, overexpression of hTERT did not seem to increase repair of mtDNA after oxidative stress, but promoted increased levels of manganese superoxide dismutase (MnSOD) and forkhead-box-protein O3 (FoxO3a) proteins during incubation in serum free medium as well as under oxidative stress, while no differences were found in protein levels of catalase. Together, our results suggest that rather than interfering with mitochondrial DNA repair mechanisms, such as BER, telomerase seems to increase antioxidant defence mechanisms to prevent mtDNA damage and to increase cellular resistance to oxidative stress. However, the result has to be reproduced in additional cellular systems in order to generalise our findings.

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Section: Discussionsupporting

confidence: 88%

Telomerase Does Not Improve DNA Repair in Mitochondria upon Stress but Increases MnSOD Protein under Serum-Free Conditions

Martens

Schmid

Akintola

et al. 2019

IJMS

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“…Treatment with ultraviolet light below 320 nm (UVB or UVC) is effective at making DNA resistant to amplification [8], but ultraviolet light that inactivates DNA also reduces the efficiency of Taq polymerase [4], [6], [9]–[11]. Application of ultrafiltration with Millipore filters (YM100) was reported to reduce Taq polymerase sensitivity in one case [12], but not in another [13].…”

Section: Introductionmentioning

confidence: 99%

Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

2009

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BackgroundPCR in principle can detect a single target molecule in a reaction mixture. Contaminating bacterial DNA in reagents creates a practical limit on the use of PCR to detect dilute bacterial DNA in environmental or public health samples. The most pernicious source of contamination is microbial DNA in DNA polymerase preparations. Importantly, all commercial Taq polymerase preparations inevitably contain contaminating microbial DNA. Removal of DNA from an enzyme preparation is problematical.Methodology/Principal FindingsThis report demonstrates that the background of contaminating DNA detected by quantitative PCR with broad host range primers can be decreased greater than 10-fold through the simple expedient of Taq enzyme dilution, without altering detection of target microbes in samples. The general method is: For any thermostable polymerase used for high-sensitivity detection, do a dilution series of the polymerase crossed with a dilution series of DNA or bacteria that work well with the test primers. For further work use the concentration of polymerase that gave the least signal in its negative control (H2O) while also not changing the threshold cycle for dilutions of spiked DNA or bacteria compared to higher concentrations of Taq polymerase.Conclusions/SignificanceIt is clear from the studies shown in this report that a straightforward procedure of optimizing the Taq polymerase concentration achieved “treatment-free” attenuation of interference by contaminating bacterial DNA in Taq polymerase preparations. This procedure should facilitate detection and quantification with broad host range primers of a small number of bona fide bacteria (as few as one) in a sample.

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“…For example, a given microbiome DNA sequence should be interpreted differently depending on whether the corresponding microbe was metabolically active, but DNA sequences alone do not provide information on whether identified sequences come from organisms that were metabolically active, quiescent, or dead. DNA sequencing can be supported by methods that discriminate microbial viability at the time of sampling [53] and the intact nature [54] of target DNA, whilst certain VOCs are consequent to microbial metabolism [44]. However, these approaches are not always sensitive and are not likely to be robust across the range of microbial and environmental diversity.…”

Section: Discussionmentioning

confidence: 99%

Microwave detection and quantification of water hidden in and on building materials: implications for healthy buildings and microbiome studies

Horsley

Thaler

2019

BMC Infect Dis

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BackgroundExcess water in all its forms (moisture, dampness, hidden water) in buildings negatively impacts occupant health but is hard to reliably detect and quantify. Recent advances in through-wall imaging recommend microwaves as a tool with a high potential to noninvasively detect and quantify water throughout buildings.MethodsMicrowaves in both transmission and reflection (radar) modes were used to perform a simple demonstration of the detection of water both on and hidden within building materials.ResultsWe used both transmission and reflection modes to detect as little as 1 mL of water between two 7 cm thicknesses of concrete. The reflection mode was also used to detect 1 mL of water on a metal surface. We observed oscillations in transmitted and reflected microwave amplitude as a function of microwave wavelength and water layer thickness, which we attribute to thin-film interference effects.ConclusionsImproving the detection of water in buildings could help design, maintenance, and remediation become more efficient and effective and perhaps increase the value of microbiome sequence data. Microwave characterization of all forms of water throughout buildings is possible; its practical development would require new collaborations among microwave physicists or engineers, architects, building engineers, remediation practitioners, epidemiologists, and microbiologists.

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Tests of the Single-hit DNA Damage Model

Cited by 9 publications

References 49 publications

Telomerase Does Not Improve DNA Repair in Mitochondria upon Stress but Increases MnSOD Protein under Serum-Free Conditions

Telomerase Does Not Improve DNA Repair in Mitochondria upon Stress but Increases MnSOD Protein under Serum-Free Conditions

Optimizing Taq Polymerase Concentration for Improved Signal-to-Noise in the Broad Range Detection of Low Abundance Bacteria

Microwave detection and quantification of water hidden in and on building materials: implications for healthy buildings and microbiome studies

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