Cell cycle regulation as a mechanism for functional separation of the apparently redundant uracil DNA glycosylases TDG and UNG2

Human Thymine-DNA Glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily. It excises uracil, thymine and a number of chemical base lesions when mispaired with guanine in double-stranded DNA. These activities are not unique to TDG; at least three additional proteins with similar enzymatic properties are present in mammalian cells. The successful co-evolution of these enzymes implies the existence of non-redundant biological functions that must be coordinated. Here, we report cell cycle regulation as a mechanism for the functional separation of apparently redundant DNA glycosylases. We show that cells entering S-phase eliminate TDG through the ubiquitin-proteasome system and then maintain a TDG-free condition until G2. Incomplete degradation of ectopically expressed TDG impedes S-phase progression and cell proliferation. The mode of cell cycle regulation of TDG is strictly inverse to that of UNG2, which peaks in and throughout S-phase and then declines to undetectable levels until it appears again just before the next S-phase. Thus, TDG- and UNG2-dependent base excision repair alternates throughout the cell cycle, and the ubiquitin-proteasome pathway constitutes the underlying regulatory syste

Cell cycle regulation as a mechanism for functional separation of the apparently redundant uracil DNA glycosylases TDG and UNG2

Human Thymine-DNA Glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily. It excises uracil, thymine and a number of chemical base lesions when mispaired with guanine in double-stranded DNA. These activities are not unique to TDG; at least three additional proteins with similar enzymatic properties are present in mammalian cells. The successful co-evolution of these enzymes implies the existence of non-redundant biological functions that must be coordinated. Here, we report cell cycle regulation as a mechanism for the functional separation of apparently redundant DNA glycosylases. We show that cells entering S-phase eliminate TDG through the ubiquitin–proteasome system and then maintain a TDG-free condition until G2. Incomplete degradation of ectopically expressed TDG impedes S-phase progression and cell proliferation. The mode of cell cycle regulation of TDG is strictly inverse to that of UNG2, which peaks in and throughout S-phase and then declines to undetectable levels until it appears again just before the next S-phase. Thus, TDG- and UNG2-dependent base excision repair alternates throughout the cell cycle, and the ubiquitin–proteasome pathway constitutes the underlying regulatory system

Insights into genotoxic effects of electromagnetic fields

The increasing use of appliances, which generate electromagnetic fields (EMFs), has provoked public concern about their safety. Scientific research into possible health effects however produced conflicting results. One of the open questions is whether or not EMF exposure has genotoxic effects. Therefore, the main objective of my thesis was to investigate DNA damage formation and repair, cell cycle progression, apoptosis and DNA damage signalling in cultured human cells under EMF exposure. In particular, the nature of possible genotoxic effects and the mechanisms underlying the cellular responses were to be addressed. As in the past, genotoxic effects of EMF exposure often could not be reproduced in independent studies, I first aimed at the validation of results of previous studies [1-3]. In these studies, different genotoxicity tests revealed increased DNA damage after exposure of human fibroblast cells to EMFs in the low frequency range, as used in power lines, as well as in the radiofrequency range, as applied by mobile phones and wireless technologies. I could show that genotoxic effects of 50Hz EMFs can be reproduced independently. Effects of radiofrequency EMF exposure, however, were detectable only in one particular cell line (HR- 1d), but not in the cell line used in the original study (ES-1). Because the visual scoring method of DNA fragmentation analysis (comet assay) used in the previous studies was criticized in the scientific community, I compared this method with an automated computerized comet analysis. This established that increases of DNA fragmentation following EMF exposure are detectable in both types of analyses. Expanding the study to other cell lines, I was able to show, that 50Hz EMF exposure in two different fibroblast cell lines but not in the cancer cell line HeLa lead to comet assay effects. Furthermore, I showed that DNA fragmentation is not found in G1 blocked cells, suggesting replicating cells to be involved in EMF directed effects. This indicated, that the DNA fragmentation detected following EMF exposure might not reflect direct induction of DNA damage but rather an EMF dependent alteration of the S-phase population. Furthermore apoptosis was suggested as confounder for comet assay effects before. Addressing this question, I found decreased replication efficiency and an increased apoptotic fraction after 50Hz EMF exposure in the fibroblast cell line showing the higher comet assay effect. Therefore, I conclude that these cells encounter problems in entering S-phase or progression through S-phase, which could lead to apoptosis and, hence, apoptotic DNA fragmentation, in a subpopulation. These effects, however, cannot entirely explain the genotoxicity observed, as the fraction of cells with increased DNA fragmentation was higher than the proportion of apoptotic cells. I then addressed the type of possible DNA damage generated by EMF exposure. An inhibitor of the DNA single strand break (SSB) sensor poly-ADP-ribosylation polymerase was used to examine an engagement of DNA single strand break repair following EMF exposure. The results showed that the increase of DNA fragmentation did not change further by applying both inhibitor and EMF exposure compared to inhibitor or EMF exposure alone. Therefore the effects appear to be epistatic, indicating that EMF exposure may affect DNA SSB repair rather than inducing DNA damage itself. To address the occurrence of DNA double strand breaks or stalled replication forks, I made use of phosphorylated H2AX (γH2AX) as a marker in EMF exposed cells. This revealed no difference between non-exposed and exposed cells, suggesting that the increase in DNA fragmentation is unlikely due to such lesions. Biological effects of EMF exposure were hypothesized to reflect an influence on the free radical pool of cells and, thus oxidative stress. I examined the steady state levels of oxidative DNA damage after EMF exposure and found no indications for increased generation of indicator lesions. This result fails to support the hypothesis of EMF induced oxidative stress, although I cannot completely rule out small changes of a sub-detectable level. DNA base excision repair (BER) is the system specifically repairing small lesions including oxidative DNA base damage. To examine, if this pathway is activated during EMF exposure, I examined the formation and levels of nuclear XRCC1 foci. XRCC1 is a central component of the BER system and can be seen to localize to sites of DNA damage and repair. However, immunostaining of XRCC1 revealed no difference in numbers and distribution of foci following EMF exposure. Adding a DNA Polymerase β (the BER polymerase) inhibitor, however, the subG1 fraction of cells increased synergistically with ELF-EMF exposure. This could indicate, that either BER protects cells from entering apoptosis following EMF exposure or that the DNA damage generated by inhibiting DNA Polymerase β is less efficiently processed under EMF exposure. Taken together, these results suggest, that the small increase in DNA fragmentation observed in human fibroblasts exposed to 50Hz EMFs can be accounted for by a combination of effects including impaired repair of endogenously arising DNA damage, disturbance of Sphase progression and apoptosis in a small fraction of cells, rather than by directly induced DNA damage. In a second part of my thesis, I used the highly sensitive comet assay, cell cycle analysis and immunofluorescence staining technologies established for the EMF studies to contribute to different projects addressing regulatory aspects of DNA BER. In a first study, we showed that Thymine DNA Glycosylase (TDG) levels were cell cycle regulated and TDG is absent in Sphase in biochemical assays. Regulation occurs at the protein level, as mRNA levels remain constant throughout the cell cycle. The protein is ubiquitinated and degraded by the proteasome. To provide biological evidence for such a regulation in vivo, I stained cells with antibodies for TDG and the S-phase marker PCNA by immunofluorescence and counted cell numbers of double and single stained cells. PCNA positive cells did not stain for TDG and vice versa. As PCNA is a marker for S-phase, this shows, that TDG is absent in S-phase cells. In a second study we provided evidence for a regulation of DNA Polymerase β (DNA Pol β) by protein arginine methylation. This methylation has impact on its in vitro performance like DNA binding and processivity, but an in vivo relevance of this modification remained to be shown. I showed that DNA Pol β knock out cells complemented with a mutated form of DNA Pol β, not able to be methylated, showed a higher level of DNA fragmentation upon induced DNA damage than cell complemented with wild type DNA Pol β. Together with reduced survival rates and an increased subG1 fraction in cells challenged with a DNA damaging agent, this established the in vivo relevance of DNA Pol β methylation. Arginine methylation therefore might represent a novel regulatory protein modification in DNA BER. In a third study, I contributed to the investigation of the toxicity mechanism of the chemotherapeutic drug 5-fluorouracil (5-FU), which is not fully understood so far. An involvement of the BER enzyme TDG was suggested by biochemical evidence, leading to the question, if TDG wild type and knock out cells respond differently to 5-FU. TDG knock out cells displayed hypersensitivity to 5-FU, which suggested a deleterious repair mechanism through TDG, probably leading to the induction of DNA SSBs. I indeed found increased DNA strand breaks in TDG wild type cells compared to knock out cells, while XRCC1, a marker for BER, was more activated in knock out cells. In cell cycle analyses 5-FU induced accumulation on S-phase of TDG deficient cells was less pronounced than in wild type cells. This suggests that TDG contributes to 5-FU mediated cytotoxicity, probably by inducing DNA SSBs due to its slow turnover rate and the resulting saturation of BER, leading then to checkpoint activation and S-phase accumulation

Base excision by thymine DNA glycosylase mediates DNA-directed cytotoxicity of 5-fluorouracil

5-Fluorouracil (5-FU), a chemotherapeutic drug commonly used in cancer treatment, imbalances nucleotide pools, thereby favoring misincorporation of uracil and 5-FU into genomic DNA. The processing of these bases by DNA repair activities was proposed to cause DNA-directed cytotoxicity, but the underlying mechanisms have not been resolved. In this study, we investigated a possible role of thymine DNA glycosylase (TDG), one of four mammalian uracil DNA glycosylases (UDGs), in the cellular response to 5-FU. Using genetic and biochemical tools, we found that inactivation of TDG significantly increases resistance of both mouse and human cancer cells towards 5-FU. We show that excision of DNA-incorporated 5-FU by TDG generates persistent DNA strand breaks, delays S-phase progression, and activates DNA damage signaling, and that the repair of 5-FU-induced DNA strand breaks is more efficient in the absence of TDG. Hence, excision of 5-FU by TDG, but not by other UDGs (UNG2 and SMUG1), prevents efficient downstream processing of the repair intermediate, thereby mediating DNA-directed cytotoxicity. The status of TDG expression in a cancer is therefore likely to determine its response to 5-FU-based chemotherapy

Assessment of Genotoxicity in Human Cells Exposed to Modulated Electromagnetic Fields of Wireless Communication Devices

Modulated electromagnetic fields (wEMFs), as generated by modern communication technologies, have raised concerns about adverse health effects. The International Agency for Research on Cancer (IARC) classifies them as “possibly carcinogenic to humans” (Group 2B), yet, the underlying molecular mechanisms initiating and promoting tumorigenesis remain elusive. Here, we comprehensively assess the impact of technologically relevant wEMF modulations on the genome integrity of cultured human cells, investigating cell type-specificities as well as time- and dose-dependencies. Classical and advanced methodologies of genetic toxicology and DNA repair were applied, and key experiments were performed in two separate laboratories. Overall, we found no conclusive evidence for an induction of DNA damage nor for alterations of the DNA repair capacity in cells exposed to several wEMF modulations (i.e., GSM, UMTS, WiFi, and RFID). Previously reported observations of increased DNA damage after exposure of cells to GSM-modulated signals could not be reproduced. Experimental variables, presumably underlying the discrepant observations, were investigated and are discussed. On the basis of our data, we conclude that the possible carcinogenicity of wEMF modulations cannot be explained by an effect on genome integrity through direct DNA damage. However, we cannot exclude non-genotoxic, indirect, or secondary effects of wEMF exposure that may promote tumorigenesis in other ways

Base Excision by Thymine DNA Glycosylase Mediates DNA-Directed Cytotoxicity of 5-Fluorouracil

5-Fluorouracil (5-FU), a chemotherapeutic drug commonly used in cancer treatment, imbalances nucleotide pools, thereby favoring misincorporation of uracil and 5-FU into genomic DNA. The processing of these bases by DNA repair activities was proposed to cause DNA-directed cytotoxicity, but the underlying mechanisms have not been resolved. In this study, we investigated a possible role of thymine DNA glycosylase (TDG), one of four mammalian uracil DNA glycosylases (UDGs), in the cellular response to 5-FU. Using genetic and biochemical tools, we found that inactivation of TDG significantly increases resistance of both mouse and human cancer cells towards 5-FU. We show that excision of DNA-incorporated 5-FU by TDG generates persistent DNA strand breaks, delays S-phase progression, and activates DNA damage signaling, and that the repair of 5-FU–induced DNA strand breaks is more efficient in the absence of TDG. Hence, excision of 5-FU by TDG, but not by other UDGs (UNG2 and SMUG1), prevents efficient downstream processing of the repair intermediate, thereby mediating DNA-directed cytotoxicity. The status of TDG expression in a cancer is therefore likely to determine its response to 5-FU–based chemotherapy

Arginine methylation regulates DNA polymerase beta

Alterations in DNA repair lead to genomic instability and higher risk of cancer. DNA base excision repair (BER) corrects damaged bases, apurinic sites, and single-strand DNA breaks. Here, a regulatory mechanism for DNA polymerase beta (Pol beta) is described. Pol beta was found to form a complex with the protein arginine methyltransferase 6 (PRMT6) and was specifically methylated in vitro and in vivo. Methylation of Pol beta by PRMT6 strongly stimulated DNA polymerase activity by enhancing DNA binding and processivity, while single nucleotide insertion and dRP-lyase activity were not affected. Two residues, R83 and R152, were identified in Pol beta as the sites of methylation by PRMT6. Genetic complementation of Pol beta knockout cells with R83/152K mutant revealed the importance of these residues for the cellular resistance to DNA alkylating agent. Based on our findings, we propose that PRMT6 plays a role as a regulator of BER