A Genetic Map of the Response to DNA Damage in Human Cells

Effects of Maillard reaction products on DNA damage in human cells and their possible mechanisms

A novel function of emodin: Enhancement of the nucleotide excision repair of UV- and cisplatin-induced DNA damage in human cells

Mutations caused by ultraviolet (UV)-induced DNA damage represent the initial genetic changes in the tumorigenesis of UV-induced skin cancer. Different wavelengths of UV radiation cause different kinds of DNA damage and mutations. UVB (290-320 nm) generates pyrimidine dimers by direct excitation of the DNA molecule. UVA (320-400 nm) can damage the DNA only indirectly through a photosensitized reaction. This indirect action is mediated mainly by singlet oxygen, which generates purine base modifications, and has been implicated in the carcinogenic effects of UVA. In order to study the processing of directly and indirectly UV-induced DNA damage in human cells, we first treated the replicating plasmid pRSVcat with up to 10 kJ/m2 UVB or with the photosensitizer methylene blue plus visible light (which generates singlet oxygen) in vitro. Then, the damaged plasmid was transfected into normal or repair deficient xeroderma pigmentosum complementation group A (XP-A) cells. DNA repair was assessed by measuring activity of reactivated chloramphenicol acetyltransferase (CAT) enzyme, encoded by the plasmid's cat gene, in cell extracts after 3 days. While XP-A cells exhibited a significantly reduced repair of UVB-induced DNA damage, they showed a normal repair of singlet oxygen-induced DNA damage. This indicates a differential DNA repair pathway for directly and indirectly UV-induced DNA damage in human cells. Irradiation of the plasmid with UVA alone did not result in a genotoxic effect. Only in conjunction with a cell extract, which provides all candidate cellular photosensitizers, did we find a reduced CAT activity after transfection. This indicates that the genotoxicity of UVA is mediated by a cellular photosensitizer.

Methyl methanesulfonate (MMS) induces mutations in a nonlinear (hockey-stick-shaped) dose-response curve in DT40 cells, about 55% of which are located at A:T sites with a high frequency of A to T mutations. We hypothesized that these transversion mutations are due to base excision repair (BER) intermediates, such as abasic sites and single strand breaks derived from N3-methyladenine. One candidate polymerase to bypass abasic sites is polymerase delta (POLD). POLD is responsible for the lagging strand during DNA replication. POLD is referred to as a high fidelity DNA polymerase because of its ability to discriminate deoxynucleotides and its 3’->5’ exo-nuclease activity. One of the POLD subunits, POLD3, is important to recruit PCNA binding to the POLD core enzyme and is implied to have critical roles for translesion DNA synthesis. In yeast, cells deficient in POLD3 homologous gene POL32 are sensitive to DNA damaging agents and resistant to mutations caused by mutagens, such as MMS and UV. Here we generated POLD3 knock-down (KD) cells via lentivirus based shRNA in three different human cell lines. All POLD3 KD cells grow slower than the control cells, suggesting POLD3 function tolerates endogenous DNA lesions. Furthermore, we found that POLD3 KD cells were sensitive to various genotoxic agents including MMS and potassium chromate. To further investigate the role of POLD3 in the DNA damage response, we introduced a series of deletion mutations of POLD3 in POLD3 deficient cells. We found that both the POLD2 binding domain and the middle linker domain are critical for the activity of POLD3 while the C-terminal PCNA binding domain is dispensable. While there is little homology between human POLD3 and yeast POL32, function of these two proteins appears to be quite similar in structure biology analysis. Our results also suggest that POLD3 plays important roles to bypass DNA lesions caused by endogenous and various exogenous genotoxic agents. Citation Format: Xu Tian, James Swenberg, Jun Nakamura. POLD3 is required for DNA damage response to endogenous and exogenous DNA damage in human cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1281. doi:10.1158/1538-7445.AM2013-1281

Recombinant adenoviral vectors provide efficient means for gene transduction in mammalian cells in vitro and in vivo. We are currently using these vectors to transduce DNA repair genes into repair deficient cells, derived from xeroderma pigmentosum (XP) patients. XP is an autosomal syndrome characterized by a high frequency of skin tumors, especially in areas exposed to sunlight, and, occasionally, developmental and neurological abnormalities. XP cells are deficient in nucleotide excision repair (affecting one of the seven known XP genes, xpa to xpg) or in DNA replication of DNA lesions (affecting DNA polymerase eta, xpv). The adenovirus approach allows the investigation of different consequences of DNA lesions in cell genomes. Adenoviral vectors carrying several xp and photolyases genes have been constructed and successfully tested in cell culture systems and in vivo directly in the skin of knockout model mice. This review summarizes these recent data and proposes the use of recombinant adenoviruses as tools to investigate the mechanisms that provide protection against DNA damage in human cells, as well as to better understand the higher predisposition of XP patients to cancer.

Investigating the efficacy of UVSE protein at repairing CPD and 6–4 pp DNA damages in human cells

Mismatch repair (MMR) proteins contribute to genome stability by excising DNA mismatches introduced by DNA polymerase. Although MMR proteins are also known to influence cellular responses to DNA damage, how MMR proteins respond to DNA damage within the cell remains unknown. Here, we show that MMR proteins are recruited immediately to the sites of various types of DNA damage in human cells. MMR proteins are recruited to single-strand breaks in a poly(ADP-ribose)-dependent manner as well as to double-strand breaks. Using mutant cells, RNA interference and expression of fluorescence-tagged proteins, we show that accumulation of MutSbeta at the DNA damage site is solely dependent on the PCNA-binding domain of MSH3, and that of MutSalpha depends on a region near the PCNA-binding domain of MSH6. MSH2 is recruited to the DNA damage site through interactions with either MSH3 or MSH6, and is required for recruitment of MLH1 to the damage site. We found, furthermore, that MutSbeta is also recruited to UV-irradiated sites in nucleotide-excision-repair- and PCNA-dependent manners. Thus, MMR and its proteins function not only in replication but also in DNA repair.

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The ‘CometChip’ is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The ‘CometChip’ is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

Antioxidant micronutrients may account for the beneficial effects of fruits on human health. A direct demonstration that consumption of fruit decreases oxidative DNA damage in human cells would support this hypothesis. Kiwifruit was taken as an example of a food with putative antioxidant properties, and its effectiveness at decreasing oxidative DNA damage was assessed in ex vivo as well as in vitro tests. The comet assay (single-cell gel electrophoresis) was used to measure DNA damage in lymphocytes collected during a human supplementation trial with a single 0.5-liter drink of kiwifruit juice (with water as a control). The comet assay was also modified to assess the antioxidant effect of kiwifruit in vitro by measuring the ability of an extract to interfere with oxidative damage to DNA induced by H2 O2. Ex vivo, consumption of kiwifruit led to an increased resistance of DNA to oxidative damage induced by H2 O2 in isolated lymphocytes, in comparison with lymphocytes collected after a control drink of water. No effect was seen on endogenous DNA damage. In vitro, a simple extract of kiwifruit, buffered to pH 7, was more effective than a solution of vitamin C (of equivalent concentration) at protecting DNA from damage, whereas at the highest concentrations tested, neither kiwi extract nor vitamin C had a protective effect. We have demonstrated significant antioxidant activity of kiwifruit ex vivo and in vitro, not attributable entirely to the vitamin C content of the fruit. Our dual approach is appropriate for testing other fruit and vegetable products for potential antioxidant effects.

The tumor suppressor p531 is a master regulator of cell cycle checkpoint responses to DNA damage whose activity is tightly controlled. It functions as a transcription factor for genes regulating cell cycle progression and apoptotic cell death. Following DNA damage, p53 is stabilized and its transcriptional activity is markedly stimulated. Among the many factors that can modulate p53 activity, the RNA-binding protein hnRNP K (heterogeneous ribonucleoprotein K) was characterized as a key player in the p53-dependent response to DNA damage in human cells, acting as a transcriptional coactivator for p53.2 hnRNP K and p53 are indeed co-recruited to the promoters of p53-responsive genes and cooperate to elicit their activation. Interestingly, similar to p53, hnRNP K levels are regulated by the E3 ubiquitin ligase HDM2, which targets hnRNP K for ubiquitylation and proteasomal degradation.2 DNA damage triggers the dissociation of the hnRNP K-HDM2 complex, leading to hnRNP K stabilization. Because these events are dependent on the DNA damage checkpoint kinases ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related),2 it was tempting to speculate that hnRNP K function in the DDR (DNA damage response) could be regulated by phosphorylation. This issue has been explored in a recent study published in Cell Cycle, where Moumen et al. showed that a phosphorylated form of hnRNP K can be detected with a phospho-ATM/ATR substrate antibody upon IR (ionizing radiation)-induced damage in human cells.3 Given that hnRNP K levels are elevated in an ATM-dependent manner in response to IR,2 the authors tested the possibility that this key upstream DNA damage checkpoint kinase could target hnRNP K. Using an ATM-specific inhibitor and siRNA-mediated depletion of the ATM kinase, they demonstrate that hnRNP K is phosphorylated in an ATM-dependent manner after IR. They also identify four S/T-Q ATM consensus target motifs in the hnRNP K sequence (S121, T174, T390, T440) that can serve as ATM phosphorylation sites. Similar phosphorylation events may be mediated by ATR following UV irradiation, because hnRNP K is stabilized in an ATR-dependent manner in this context.2 Next, Moumen et al. examined the functional relevance of such phosphorylation of hnRNP K in the DDR. Taking advantage of a phospho-deficient mutant where the four S/T sites are mutated to alanines, they established that phosphorylation of hnRNP K is critical for its dissociation from HDM2 and subsequent stabilization in response to IR. It is not yet clear whether the ATM-dependent phosphorylation of hnRNP K directly inhibits its interaction with HDM2, because how hnRNP K interacts with HDM2 is currently unknown. Importantly, cells expressing the phospho-deficient hnRNP K protein also display impaired recruitment of p53 to its target gene p21Waf1 and defective stimulation of p53 transcriptional activity. Together, these findings support a model where ATM-mediated phosphorylation protects hnRNP K from proteasomal degradation in response to damage, allowing it function as a coactivator for p53 (Fig. 1). Identifying phosphatases able to reverse hnRNP K phosphorylation would help define how this response is switched off after damage. Figure 1. Control of hnRNP K by phosphorylation: Integrating DNA damage signals to fine-tune p53-dependent transcriptional responses. Remarkably, such phospho-dependent control of hnRNP K levels in response to DNA damage mirrors the upregulation of p53, whose phosphorylation by ATM also leads to its stabilization upon dissociation from HDM2. This striking parallel between two proteins collaborating in the DDR reveals the tight control that ATM exerts on p53 transcriptional activity. By achieving the same goal via two converging pathways, ATM imposes a double lock on cell cycle checkpoint responses (Fig. 1). These observations should be considered in light of other DNA damage-dependent modifications on hnRNP K that control p53-dependent transcription, including sumoylation and methylation.4-6 It will be of major interest to identify potential cross-talk and/or interference between these modifications to shed light on how they collectively contribute to the fine-tuning of hnRNP K activity in response to damage. It would also be important to evaluate whether ATM-dependent phosphorylation effects other functions of hnRNP K in RNA metabolism, including its ability to repress p53-target genes via an interaction with the p53-induced large intergenic noncoding RNA lincRNA-p21.7 Finally, the critical role of ATM phospho-target sites in regulating hnRNP K protein levels opens up the possibility that mutations of these residues could be instrumental in driving the upregulation of hnRNP K frequently observed in human tumors.

Translesion synthesis by DNA polymerase eta (polη) is one mechanism by which cancer cells can tolerate DNA damage by platinum-based anti-cancer drugs. Cells lacking polη are sensitive to these agents. To help define the consequences of polη-deficiency, we characterized the effects of equitoxic doses of cisplatin and carboplatin on cell cycle progression and activation of DNA damage response pathways in a human cell line lacking polη. We show that both cisplatin and carboplatin induce strong S-phase arrest in polη-deficient XP30RO cells, associated with reduced expression of cyclin E and cyclin B. PIK kinase-mediated phosphorylation of Chk1, H2AX and RPA2 was strongly activated by both cisplatin and carboplatin, but phosphorylation of these proteins was induced earlier by cisplatin than by an equitoxic dose of carboplatin. Compared to Chk1 and H2AX phosphorylation, RPA2 hyperphosphorylation on serine4/serine8 is a late event in response to platinum-induced DNA damage. We directly demonstrate, using dual-labeling flow cytometry, that damage-induced phosphorylation of RPA2 on serine4/serine8 occurs primarily in the S and G2 phases of the cell cycle, and show that the timing of RPA2 phosphorylation can be modulated by inhibition of the checkpoint kinase Chk1. Furthermore, Chk1 inhibition sensitizes polη-deficient cells to the cytotoxic effects of carboplatin. Both hyperphosphorylated RPA2 and the homologous recombination protein Rad51 are present in nuclear foci after cisplatin treatment, but these are separable events in individual cells. These results provide insight into the relationship between cell cycle regulation and processing of platinum-induced DNA damage in human cells when polη-mediated TLS is compromised.

Detection of in vivo DNA damage induced by very low doses of mainstream and sidestream smoke extracts using a novel assay.

Modulation of the DNA-damage response to HZE particles by shielding

Oxidative stress and EROD activity in Caco-2 cells upon exposure to chlorinated hydrophobic organic compounds from drinking water reservoirs