Bevin P. Engelward

Base excision repair intermediates induce p53-independent cytotoxic and genotoxic responses.

DNA alkylation damage is primarily repaired by the base excision repair (BER) machinery in mammalian cells. In repair of the N-alkylated purine base lesion, for example, alkyl adenine DNA glycosylase (Aag) recognizes and removes the base, and DNA polymerase β (β-pol) contributes the gap tailoring and DNA synthesis steps. It is the loss of β-pol-mediated 5′-deoxyribose phosphate removal that renders mouse fibroblasts alkylation-hypersensitive. Here we report that the hypersensitivity of β-pol-deficient cells after methyl methanesulfonate-induced alkylation damage is wholly dependent upon glycosylase-mediated initiation of repair, indicating that alkylated base lesions themselves are tolerated in these cells and demonstrate that β-pol protects against accumulation of toxic BER intermediates. Further, we find that these intermediates are initially tolerated in vivo by a second repair pathway, homologous recombination, inducing an increase in sister chromatid exchange events. If left unresolved, these BER intermediates trigger a rapid block in DNA synthesis and cytotoxicity. Surprisingly, both the cytotoxic and genotoxic signals are independent of both the p53 response and mismatch DNA repair pathways, demonstrating that p53 is not required for a functional BER pathway, that the observed damage response is not part of the p53 response network, and that the BER intermediate-induced cytotoxic and genotoxic effects are distinct from the mechanism engaged in response to mismatch repair signaling. These studies demonstrate that, although base damage is repaired by the BER pathway, incomplete BER intermediates are shuttled into the homologous recombination pathway, suggesting possible coordination between BER and the recombination machinery.

Single cell trapping and DNA damage analysis using microwell arrays

With a direct link to cancer, aging, and heritable diseases as well as a critical role in cancer treatment, the importance of DNA damage is well-established. The intense interest in DNA damage in applications ranging from epidemiology to drug development drives an urgent need for robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. We have developed a simple method for high throughput DNA damage measurements that provides information on multiple lesions and pathways. Our method utilizes single cells captured by gravity into a microwell array with DNA damage revealed morphologically by gel electrophoresis. Spatial encoding enables simultaneous assays of multiple experimental conditions performed in parallel with fully automated analysis. This method also enables novel functionalities, including multiplexed labeling for parallel single cell assays, as well as DNA damage measurement in cell aggregates. We have also developed 24- and 96-well versions, which are applicable to high throughput screening. Using this platform, we have quantified DNA repair capacities of individuals with different genetic backgrounds, and compared the efficacy of potential cancer chemotherapeutics as inhibitors of a critical DNA repair enzyme, human AP endonuclease. This platform enables high throughput assessment of multiple DNA repair pathways and subpathways in parallel, thus enabling new strategies for drug discovery, genotoxicity testing, and environmental health.

Recombinational Repair Is Critical for Survival of Escherichia coli Exposed to Nitric Oxide

Nitric oxide (NO(.)) is critical to numerous biological processes, including signal transduction and macrophage-mediated immunity. In this study, we have explored the biological effects of NO(.)-induced DNA damage on Escherichia coli. The relative importance of base excision repair, nucleotide excision repair (NER), and recombinational repair in preventing NO(.)-induced toxicity was determined. E. coli strains lacking either NER or DNA glycosylases (including those that repair alkylation damage [alkA tag strain], oxidative damage [fpg nei nth strain], and deaminated cytosine [ung strain]) showed essentially wild-type levels of NO(.) resistance. However, apyrimidinic/apurinic (AP) endonuclease-deficient cells (xth nfo strain) were very sensitive to killing by NO(.), which indicates that normal processing of abasic sites is critical for defense against NO(.). In addition, recA mutant cells were exquisitely sensitive to NO(.)-induced killing. Both SOS-deficient (lexA3) and Holliday junction resolvase-deficient (ruvC) cells were very sensitive to NO(.), indicating that both SOS and recombinational repair play important roles in defense against NO(.). Furthermore, strains specifically lacking double-strand end repair (recBCD strains) were very sensitive to NO(.), which suggests that NO(.) exposure leads to the formation of double-strand ends. One consequence of these double-strand ends is that NO(.) induces homologous recombination at a genetically engineered substrate. Taken together, it is now clear that, in addition to the known point mutagenic effects of NO(.), it is also important to consider recombination events among the spectrum of genetic changes that NO(. ) can induce. Furthermore, the importance of recombinational repair for cellular survival of NO(.) exposure reveals a potential susceptibility factor for invading microbes.

Base excision repair and nucleotide excision repair contribute to the removal of N-methylpurines from active genes

Many different cellular pathways have evolved to protect the genome from the deleterious effects of DNA damage that result from exposure to chemical and physical agents. Among these is a process called transcription-coupled repair (TCR) that catalyzes the removal of DNA lesions from the transcribed strand of expressed genes, often resulting in a preferential bias of damage clearance from this strand relative to its non-transcribed counterpart. Lesions subject to this type of repair include cyclobutane pyrimidine dimers that are normally repaired by nucleotide excision repair (NER) and thymine glycols (TGs) that are removed primarily by base excision repair (BER). While the mechanism underlying TCR is not completely clear, it is known that its facilitation requires proteins used by other repair pathways like NER. It is also believed that the signal for TCR is the stalled RNA polymerase that results when DNA damage prevents its translocation during transcription elongation. While there is a clear role for some NER proteins in TCR, the involvement of BER proteins is less clear. To explore this further, we studied the removal of 7-methylguanine (7MeG) and 3-methyladenine (3MeA) from the dihydrofolate reductase (dhfr) gene of murine cell lines that vary in their repair phenotypes. 7MeG and 3MeA constitute the two principal N-methylpurines formed in DNA following exposure to methylating agents. In mammalian cells, alkyladenine DNA alkyladenine glycosylase (Aag) is the major enzyme required for the repair of these lesions via BER, and their removal from the total genome is quite rapid. There is no observable TCR of these lesions in specific genes in DNA repair proficient cells; however, it is possible that the rapid repair of these adducts by BER masks any TCR. The repair of 3MeA and 7MeG was examined in cells lacking Aag, NER, or both Aag and NER to determine if rapid overall repair masks TCR. The results show that both 3MeA and 7MeG are removed without strand bias from the dhfr gene of BER deficient (Aag deficient) and NER deficient murine cell lines. Furthermore, repair of 3MeA in this region is highly dependent on Aag, but repair of 7MeG is equally efficient in the repair proficient, BER deficient, and NER deficient cell lines. Strikingly, in the absence of both BER and NER, neither 7MeG nor 3MeA is repaired. These results demonstrate that NER, but not TCR, contributes to the repair of 7MeG, and to a lesser extent 3MeA.

In vivo and in vitro studies on the roles of neutrophil extracellular traps during secondary pneumococcal pneumonia after primary pulmonary influenza infection

Seasonal influenza virus infections may lead to debilitating disease, and account for significant fatalities annually worldwide. Most of these deaths are attributed to the complications of secondary bacterial pneumonia. Evidence is accumulating to support the notion that neutrophil extracellular traps (NETs) harbor several antibacterial proteins, and trap and kill bacteria. We have previously demonstrated the induction of NETs that contribute to lung tissue injury in severe influenza pneumonia. However, the role of these NETs in secondary bacterial pneumonia is unclear. In this study, we explored whether NETs induced during pulmonary influenza infection have functional significance against infections with Streptococcus pneumoniae and other bacterial and fungal species. Our findings revealed that NETs do not participate in killing of Streptococcus pneumoniae in vivo and in vitro. Dual viral and bacterial infection elevated the bacterial load compared to animals infected with bacteria alone. Concurrently, enhanced lung pathogenesis was observed in dual-infected mice compared to those challenged with influenza virus or bacteria alone. The intensified NETs in dual-infected mice often appeared as clusters that were frequently filled with partially degraded DNA, as evidenced by punctate histone protein staining. The severe pulmonary pathology and excessive NETs generation in dual infection correlated with exaggerated inflammation and damage to the alveolar-capillary barrier. NETs stimulation in vitro did not significantly alter the gene expression of several antimicrobial proteins, and these NETs did not exhibit any bactericidal activity. Fungicidal activity against Candida albicans was observed at similar levels both in presence or absence of NETs. These results substantiate that the NETs released by primary influenza infection do not protect against secondary bacterial infection, but may compromise lung function.

Inflammation-induced cell proliferation potentiates DNA damage-induced mutations in vivo.

Mutations are a critical driver of cancer initiation. While extensive studies have focused on exposure-induced mutations, few studies have explored the importance of tissue physiology as a modulator of mutation susceptibility in vivo. Of particular interest is inflammation, a known cancer risk factor relevant to chronic inflammatory diseases and pathogen-induced inflammation. Here, we used the fluorescent yellow direct repeat (FYDR) mice that harbor a reporter to detect misalignments during homologous recombination (HR), an important class of mutations. FYDR mice were exposed to cerulein, a potent inducer of pancreatic inflammation. We show that inflammation induces DSBs (γH2AX foci) and that several days later there is an increase in cell proliferation. While isolated bouts of inflammation did not induce HR, overlap between inflammation-induced DNA damage and inflammation-induced cell proliferation induced HR significantly. To study exogenously-induced DNA damage, animals were exposed to methylnitrosourea, a model alkylating agent that creates DNA lesions relevant to both environmental exposures and cancer chemotherapy. We found that exposure to alkylation damage induces HR, and importantly, that inflammation-induced cell proliferation and alkylation induce HR in a synergistic fashion. Taken together, these results show that, during an acute bout of inflammation, there is a kinetic barrier separating DNA damage from cell proliferation that protects against mutations, and that inflammation-induced cell proliferation greatly potentiates exposure-induced mutations. These studies demonstrate a fundamental mechanism by which inflammation can act synergistically with DNA damage to induce mutations that drive cancer and cancer recurrence.

Homologous recombination prevents methylation-induced toxicity in Escherichia coli

Methylating agents such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and methyl methane sulfonate (MMS) produce a wide variety of N- and O-methylated bases in DNA, some of which can block replication fork progression. Homologous recombination is a mechanism by which chromosome replication can proceed despite the presence of lesions. The two major recombination pathways, RecBCD and RecFOR, which repair double-strand breaks (DSBs) and single-strand gaps respectively, are needed to protect against toxicity with the RecBCD system being more important. We find that recombination-deficient cell lines, such as recBCD recF, and ruvC recG, are as sensitive to the cytotoxic effects of MMS and MNNG as the most base excision repair (BER)-deficient (alkA tag) isogenic mutant strain. Recombination and BER-deficient double mutants (alkA tag recBCD) were more sensitive to MNNG and MMS than the single mutants suggesting that homologous recombination and BER play essential independent roles. Cells deleted for the polA (DNA polymerase I) or priA (primosome) genes are as sensitive to MMS and MNNG as alkA tag bacteria. Our results suggest that the mechanism of cytotoxicity by alkylating agents includes the necessity for homologous recombination to repair DSBs and single-strand gaps produced by DNA replication at blocking lesions or single-strand nicks resulting from AP-endonuclease action.

Spontaneous mitotic homologous recombination at an enhanced yellow fluorescent protein (EYFP) cDNA direct repeat in transgenic mice.

A transgenic mouse has been created that provides a powerful tool for revealing genetic and environmental factors that modulate mitotic homologous recombination. The fluorescent yellow direct-repeat (FYDR) mice described here carry two different copies of expression cassettes for truncated coding sequences of the enhanced yellow fluorescent protein (EYFP), arranged in tandem. Homologous recombination between these repeated elements can restore full-length EYFP coding sequence to yield a fluorescent phenotype, and the resulting fluorescent recombinant cells are rapidly quantifiable by flow cytometry. Analysis of genomic DNA from recombined FYDR cells shows that this mouse model detects gene conversions, and based on the arrangement of the integrated recombination substrate, unequal sister-chromatid exchanges and repair of collapsed replication forks are also expected to reconstitute EYFP coding sequence. The rate of spontaneous recombination in primary fibroblasts derived from adult ear tissue is 1.3 ± 0.1 per 106 cell divisions. Interestingly, the rate is ≈10-fold greater in fibroblasts derived from embryonic tissue. We observe an ≈15-fold increase in the frequency of recombinant cells in cultures of ear fibroblasts when exposed to mitomycin C, which is consistent with the ability of interstrand crosslinks to induce homologous recombination. In addition to studies of recombination in cultured primary cells, the frequency of recombinant cells present in skin was also measured by direct analysis of disaggregated cells. Thus, the FYDR mouse model can be used for studies of mitotic homologous recombination both in vitro and in vivo.

Three-dimensional tissue cytometer based on high-speed multiphoton microscopy.

Image cytometry technology has been extended to 3D based on high‐speed multiphoton microscopy. This technique allows in situ study of tissue specimens preserving important cell–cell and cell–extracellular matrix interactions. The imaging system was based on high‐speed multiphoton microscopy (HSMPM) for 3D deep tissue imaging with minimal photodamage. Using appropriate fluorescent labels and a specimen translation stage, we could quantify cellular and biochemical states of tissues in a high throughput manner. This approach could assay tissue structures with subcellular resolution down to a few hundred micrometers deep. Its throughput could be quantified by the rate of volume imaging: 1.45 mm3/h with high resolution. For a tissue containing tightly packed, stratified cellular layers, this rate corresponded to sampling about 200 cells/s. We characterized the performance of 3D tissue cytometer by quantifying rare cell populations in 2D and 3D specimens in vitro. The measured population ratios, which were obtained by image analysis, agreed well with the expected ratios down to the ratio of 1/105. This technology was also applied to the detection of rare skin structures based on endogenous fluorophores. Sebaceous glands and a cell cluster at the base of a hair follicle were identified. Finally, the 3D tissue cytometer was applied to detect rare cells that had undergone homologous mitotic recombination in a novel transgenic mouse model, where recombination events could result in the expression of enhanced yellow fluorescent protein in the cells. 3D tissue cytometry based on HSMPM demonstrated its screening capability with high sensitivity and showed the possibility of studying cellular and biochemical states in tissues in situ. This technique will significantly expand the scope of cytometric studies to the biomedical problems where spatial and chemical relationships between cells and their tissue environments are important.

Streptococcus pneumoniae secretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

Significance Streptococcus pneumoniae is the most common cause of pneumonia, a leading cause of death globally. Limitations in antibiotic efficacy and vaccines call attention to the need to develop our understanding of host–pathogen interactions to improve mitigation strategies. Here, we show that lung cells exposed to S. pneumoniae are subject to DNA damage caused by hydrogen peroxide, which is secreted by strains of S. pneumoniae that carry the spxB gene. The observation that S. pneumoniae secretes hydrogen peroxide at genotoxic and cytotoxic levels is consistent with a model wherein host DNA damage and repair modulate pneumococcal pathogenicity. Streptococcus pneumoniae is a leading cause of pneumonia and one of the most common causes of death globally. The impact of S. pneumoniae on host molecular processes that lead to detrimental pulmonary consequences is not fully understood. Here, we show that S. pneumoniae induces toxic DNA double-strand breaks (DSBs) in human alveolar epithelial cells, as indicated by ataxia telangiectasia mutated kinase (ATM)-dependent phosphorylation of histone H2AX and colocalization with p53-binding protein (53BP1). Furthermore, results show that DNA damage occurs in a bacterial contact-independent fashion and that Streptococcus pyruvate oxidase (SpxB), which enables synthesis of H2O2, plays a critical role in inducing DSBs. The extent of DNA damage correlates with the extent of apoptosis, and DNA damage precedes apoptosis, which is consistent with the time required for execution of apoptosis. Furthermore, addition of catalase, which neutralizes H2O2, greatly suppresses S. pneumoniae-induced DNA damage and apoptosis. Importantly, S. pneumoniae induces DSBs in the lungs of animals with acute pneumonia, and H2O2 production by S. pneumoniae in vivo contributes to its genotoxicity and virulence. One of the major DSBs repair pathways is nonhomologous end joining for which Ku70/80 is essential for repair. We find that deficiency of Ku80 causes an increase in the levels of DSBs and apoptosis, underscoring the importance of DNA repair in preventing S. pneumoniae-induced genotoxicity. Taken together, this study shows that S. pneumoniae-induced damage to the host cell genome exacerbates its toxicity and pathogenesis, making DNA repair a potentially important susceptibility factor in people who suffer from pneumonia.