Receptor-DNA interactions: EMSA and footprinting

Defining the precise promoter DNA sequence motifs where nuclear receptors and other transcription factors bind is an essential prerequisite for understanding how these proteins modulate the expression of their specific target genes. The purpose of this chapter is to provide the reader with a detailed guide with respect to the materials and the key methods required to perform this type of DNA-binding analysis. Irrespective of whether starting with purified DNA-binding proteins or somewhat crude cellular extracts, the tried-and-true procedures described here will enable one to accurately access the capacity of specific proteins to bind to DNA as well as to determine the exact sequences and DNA contact nucleotides involved. For illustrative purposes, we primarily have used the interaction of the androgen receptor with the rat probasin proximal promoter as our model system.

Instant coffee with high chlorogenic acid levels protects humans against oxidative damage of macromolecules

Scope: Coffee is among the most frequently consumed beverages. Its consumption is inversely associated to the incidence of diseases related to reactive oxygen species; the phenomenon may be due to its antioxidant properties. Our primary objective was to investigate the impact of consumption of a coffee containing high levels of chlorogenic acids on the oxidation of proteins, DNA and membrane lipids; additionally, other redox biomarkers were monitored in an intervention trial. Methods and results: The treatment group (n=36) consumed instant coffee co-extracted from green and roasted beans, whereas the control consumed water (800 mL/P/day, 5 days). A global statistical analysis of four main biomarkers selected as primary outcomes showed that the overall changes are significant. 8-Isoprostaglandin F2α in urine declined by 15.3%, 3-nitrotyrosine was decreased by 16.1%, DNA migration due to oxidized purines and pyrimidines was (not significantly) reduced in lymphocytes by 12.5 and 14.1%. Other markers such as the total antioxidant capacity were moderately increased; e.g. LDL and malondialdehyde were shifted towards a non-significant reduction. Conclusion: The oxidation of DNA, lipids and proteins associated with the incidence of various diseases and the protection against their oxidative damage may be indicative for beneficial health effects of coffee.

Exercise-induced DNA damage: Is there a relationship with inflammatory responses?

Both a systemic inflammatory response as well as DNA damage has been observed following exhaustive endurance exercise. Hypothetically, exercise-induced DNA damage might either be a consequence of inflammatory processes or causally involved in inflammation and immunological alterations after strenuous prolonged exercise (e.g. by inducing lymphocyte apoptosis and lymphocytopenia). Nevertheless, up to now only few studies have addressed this issue and there is hardly any evidence regarding a direct relationship between DNA or chromosomal damage and inflammatory responses in the context of exercise. The most conclusive picture that emerges from available data is that reactive oxygen and nitrogen species (RONS) appear to be the key effectors which link inflammation with DNA damage. Considering the time-courses of inflammatory and oxidative stress responses on the one hand and DNA effects on the other the lack of correlations between these responses might also be explained by too short observation periods. This review summarizes and discusses the recent findings on this topic. Furthermore, data from our own study are presented that aimed to verify potential associations between several endpoints of genome stability and inflammatory, immune-endocrine and muscle damage parameters in competitors of an Ironman triathlon until 19 days into recovery. The current results indicate that DNA effects in lymphocytes are not responsible for exercise-induced inflammatory responses. Furthermore, this investigation shows that inflammatory processes, vice versa, do not promote DNA damage, neither directly nor via an increased formation of RONS derived from inflammatory cells. Oxidative DNA damage might have been counteracted by training- and exercise-induced antioxidant responses. However, further studies are needed that combine advanced -omics based techniques (transcriptomics, proteomics) with state-of-the-art biochemical biomarkers to gain more insights into the underlying mechanisms.

Endurance exercise and DNA stability: Is there a link to duration and intensity?

It is commonly accepted that regular moderate intensity physical activity reduces the risk of developing many diseases. Counter intuitively, however, evidence also exists for oxidative stress resulting from acute and strenuous exercise. Enhanced formation of reactive oxygen and nitrogen species may lead to oxidatively modified lipids, proteins and nucleic acids and possibly disease. Currently, only a few studies have investigated the influence of exercise on DNA stability and damage with conflicting results, small study groups and the use of different sample matrices or methods and result units. This is the first review to address the effect of exercise of various intensities and durations on DNA stability, focusing on human population studies. Furthermore, this article describes the principles and limitations of commonly used methods for the assessment of oxidatively modified DNA and DNA stability. This review is structured according to the type of exercise conducted (field or laboratory based) and the intensity performed (i.e. competitive ultra/endurance exercise or maximal tests until exhaustion). The findings presented here suggest that competitive ultra-endurance exercise (>4h) does not induce persistent DNA damage. However, when considering the effects of endurance exercise (<4h), no clear conclusions could be drawn. Laboratory studies have shown equivocal results (increased or no oxidative stress) after endurance or exhaustive exercise. To clarify which components of exercise participation (i.e. duration, intensity and training status of subjects) have an impact on DNA stability and damage, additional carefully designed studies combining the measurement of DNA damage, gene expression and DNA repair mechanisms before, during and after exercise of differing intensities and durations are required.

No acute and persistent DNA damage after an Ironman triathlon

During acute and strenuous exercise, the enhanced formation of reactive oxygen species can induce damage to lipids, proteins, and nucleic acids. The aim of this study was to investigate the effect of an Ironman triathlon (3.8 km swim, 180 km cycle, 42 km run), as a prototype of ultra-endurance exercise, on DNA stability. As biomarkers of genomic instability, the number of micronuclei, nucleoplasmic bridges, and nuclear buds were measured within the cytokinesis-block micronucleus cytome assay in once-divided peripheral lymphocytes of 20 male triathletes. Blood samples were taken 2 days before, within 20 min after the race, and 5 and 19 days post-race. Overall, the number of micronuclei decreased (P < 0.05) after the race, remained at a low level until 5 days post-race, and declined further to 19 days post-race (P < 0.01). The frequency of nucleoplasmic bridges and nuclear buds did not change immediately after the triathlon. The number of nucleoplasmic bridge declined from 2 days pre-race to 19 days post-exercise (P < 0.05). The frequency of nuclear buds increased after the triathlon, peaking 5 days post-race (P < 0.01) and decreased to basic levels 19 days after the race (P < 0.01). The results suggest that an Ironman triathlon does not cause long-lasting DNA damage in well-trained athletes.

DNA damage in response to an Ironman triathlon

The major aims of this study were to investigate the effect of an Ironman triathlon on DNA migration in the single cell gel electrophoresis assay, apoptosis and necrosis in the cytokinesis-block micronucleus cytome assay with lymphocytes and on changes of total antioxidant capacity in plasma. Blood samples were taken 2 days (d) before, within 20 min, 1 d, 5 d and 19 d post-race. The level of strand breaks decreased (p<0.05) immediately after the race, then increased (p<0.01) 1 d post-race and declined (p<0.01) until 19 d post-race. Apoptotic and necrotic cells decreased (p<0.01) and the total antioxidant status increased (p<0.01) immediately after the race. The results indicate that ultra-endurance exercise does not cause prolonged DNA damage in well-trained male athletes.

Well-trained, healthy triathletes experience no adverse health risks regarding oxidative stress and DNA damage by participating in an ultra-endurance event

Also physical exercise in general is accepted to be protective, acute and strenuous exercise has been shown to induce oxidative stress. Enhanced formation of free radicals leads to oxidation of macromolecules and to DNA damage. On the other hand ultra-endurance events which require strenuous exercise are very popular and the number of participants is continuously increasing worldwide. Since only few data exists on Ironman triathletes, who are prototypes of ultra-endurance athletes, this study was aimed at assessing the risk of oxidative stress and DNA damage after finishing a triathlon and to predict a possible health risk. Blood samples of 42 male athletes were taken 2 days before, within 20 min after the race, 1, 5 and 19 days post-race. Oxidative stress marker increased only moderately after the race and returned to baseline after 5 days. Marker of DNA damage measured by the SCGE assay with and without restriction enzymes as well as by the sister chromatid exchange assay did either show no change or deceased within the first day after the race. Due to intake during the race and the release by the cells plasma concentrations of vitamin C and α-tocopherol increased after the event and returned to baseline 1 day after. This study indicates that despite a temporary increase in some oxidative stress markers, there is no persistent oxidative stress and no DNA damage in response to an Ironman triathlon in trained athletes, mainly due to an appropriate antioxidant intake and general protective alterations in the antioxidant defence system.

Impact of endurance and ultraendurance exercise on DNA damage

Regular moderate physical activity reduces the risk of several noncommunicable diseases. At the same time, evidence exists for oxidative stress resulting from acute and strenuous exercise by enhanced formation of reactive oxygen and nitrogen species, which may lead to oxidatively modified lipids, proteins, and possibly negative effects on DNA stability. The limited data on ultraendurance events such as an Ironman triathlon show no persistent DNA damage after the events. However, when considering the effects of endurance exercise comparable to a (half) marathon or a short triathlon distance, no clear conclusions could be drawn. In order to clarify which components of exercise participation, such as duration, intensity, frequency, or training status of the subjects, have an impact on DNA stability, more information is clearly needed that combines the measurement of DNA damage, gene expression, and DNA repair mechanisms before, during, and after exercise of differing intensities and durations.

Super DNAging - New insights into DNA integrity, genome stability and telomeres in the oldest old

Reductions in DNA integrity, genome stability, and telomere length are strongly associated with the aging process, age-related diseases as well as the age-related loss of muscle mass. However, in people reaching an age far beyond their statistical life expectancy the prevalence of diseases, such as cancer, cardiovascular disease, diabetes or dementia, is much lower compared to “averagely” aged humans. These inverse observations in nonagenarians (90–99 years), centenarians (100–109 years) and super-centenarians (110 years and older) require a closer look into dynamics underlying DNA damage within the oldest old of our society. Available data indicate improved DNA repair and antioxidant defense mechanisms in “super old” humans, which are comparable with much younger cohorts. Partly as a result of these enhanced endogenous repair and protective mechanisms, the oldest old humans appear to cope better with risk factors for DNA damage over their lifetime compared to subjects whose lifespan coincides with the statistical life expectancy. This model is supported by study results demonstrating superior chromosomal stability, telomere dynamics and DNA integrity in “successful agers”. There is also compelling evidence suggesting that life-style related factors including regular physical activity, a well-balanced diet and minimized psycho-social stress can reduce DNA damage and improve chromosomal stability. The most conclusive picture that emerges from reviewing the literature is that reaching “super old” age appears to be primarily determined by hereditary/genetic factors, while a healthy lifestyle additionally contributes to achieving the individual maximum lifespan in humans. More research is required in this rapidly growing population of super old people. In particular, there is need for more comprehensive investigations including short- and long-term lifestyle interventions as well as investigations focusing on the mechanisms causing DNA damage, mutations, and telomere shortening.

TMIO-pyrimid hybrids are profluorescent, site-directed spin labels for nucleic acids

We report the synthesis of a new class of molecules which are hybrids of long-lived tetramethylisoindolinoxyl (TMIO) radicals and the pyrido[1,2-a]benzimidazole (PyrImid) scaffold. These compounds represent a new lead for noncovalently binding nucleic acid probes, as they interact with nucleic acids with previously unreported C (DNA) and C/U (RNA) complementarity, which can be detected by electron paramagnetic resonance (EPR) techniques. They also have promising properties for fluorimetric analysis, as their fluorescent spin-quenched derivatives exhibit a significant Stokes shift

Biochemical profiling of proteins and metabolites in wound exudate from chronic wound environments

The lack of fundamental knowledge on the biological processes associated with wound healing represents a significant challenge. Understanding the biochemical changes that occur within a chronic wound could provide insights into the wound environment and enable more effective wound management. We report on the stability of wound fluid samples under various conditions and describe a high-throughput approach to investigate the altered biochemical state within wound samples collected from various types of chronic, ulcerated wounds. Furthermore, we discuss the viability of this approach in the early stages of wound sample protein and metabolite profiling and subsequent biomarker discovery. This approach will facilitate the detection of factors that may correlate with wound severity and/or could be used to monitor the response to a particular treatment.

Fasting increases serum bilirubin levels in clinically normal, healthy males but not females : a retrospective study from phase I clinical trial participants

Aim: To examine if fasting affects serum bilirubin levels in clinical healthy males and females. Methods: We utilised retrospective data from phase 1 clinical trials where blood was collected in either a fed or fasting state at screening and pre-dosing time points and analysed for total bilirubin levels as per standard clinical procedures. Participants were clinically healthy males (n = 105) or females (n = 30) aged 18 to 48 inclusive who participated in a phase 1 clinical trial in 2012 or 2013. Results: We found a statistically significant increase in total serum bilirubin levels in fasting males as compared to non-fasting males. The fasting time correlated positively with increased bilirubin levels. The age of the healthy males did not correlate with their fasting bilirubin level. We found no correlation between fasting and bilirubin levels in clinically normal females. Conclusions: The recruitment and screening of volunteers for a clinical trial is a time-consuming and expensive process. This study clearly demonstrates that testing for serum bilirubin should be conducted on non-fasting male subjects. If fasting is required, then participants should not be excluded from a trial based on an elevated serum bilirubin that is deemed non-clinically significant.

Involvement of Exo1b in DNA damage-induced apoptosis

Apoptosis is essential for the maintenance of inherited genomic integrity. During DNA damage-induced apoptosis, mechanisms of cell survival, such as DNA repair are inactivated to allow cell death to proceed. Here, we describe a role for the mammalian DNA repair enzyme Exonuclease 1 (Exo1) in DNA damage-induced apoptosis. Depletion of Exo1 in human fibroblasts, or mouse embryonic fibroblasts led to a delay in DNA damage-induced apoptosis. Furthermore, we show that Exo1 acts upstream of caspase-3, DNA fragmentation and cytochrome c release. In addition, induction of apoptosis with DNA-damaging agents led to cleavage of both isoforms of Exo1. The cleavage of Exo1 was mapped to Asp514, and shown to be mediated by caspase-3. Expression of a caspase-3 cleavage site mutant form of Exo1, Asp514Ala, prevented formation of the previously observed fragment without any affect on the onset of apoptosis. We conclude that Exo1 has a role in the timely induction of apoptosis and that it is subsequently cleaved and degraded during apoptosis, potentially inhibiting DNA damage repair.

Conformational flexibility revealed by the crystal structure of a crenarchaeal RadA

Homologous recombinational repair is an essential mechanism for repair of double-strand breaks in DNA. Recombinases of the RecA-fold family play a crucial role in this process, forming filaments that utilize ATP to mediate their interactions with singleand double-stranded DNA. The recombinase molecules present in the archaea (RadA) and eukaryota (Rad51) are more closely related to each other than to their bacterial counterpart (RecA) and, as a result, RadA makes a suitable model for the eukaryotic system. The crystal structure of Sulfolobus solfataricus RadA has been solved to a resolution of 3.2 A° in the absence of nucleotide analogues or DNA, revealing a narrow filamentous assembly with three molecules per helical turn. As observed in other RecA-family recombinases, each RadA molecule in the filament is linked to its neighbour via interactions of a short b-strand with the neighbouring ATPase domain. However, despite apparent flexibility between domains, comparison with other structures indicates conservation of a number of key interactions that introduce rigidity to the system, allowing allosteric control of the filament by interaction with ATP. Additional analysis reveals that the interaction specificity of the five human Rad51 paralogues can be predicted using a simple model based on the RadA structure.

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.