UV-induced Mutations Research Articles

Involvement of transcription-coupled repair factor Mfd and DNA helicase UvrD in mutational processes in Pseudomonas putida

Stalled RNA polymerases (RNAPs) pose an obstacle for the replicating complexes, which could lead to transcription-replication conflicts and result in genetic instability. Stalled RNAPs and DNA lesions blocking RNAP elongation are removed by transcription-coupled repair (TCR), the process which in bacteria is mediated by TCR factor Mfd and helicase UvrD. Although the mechanism of TCR has been extensively studied, its role in mutagenesis is still obscure. In the current study we have investigated the role of Mfd and UvrD in mutational processes in soil bacterium Pseudomonas putida. Our results revealed that UvrD helicase is essential to prevent the emergence of mutations, as the loss of uvrD resulted in elevated mutant frequency both in exponential- and stationary-phase bacterial cultures. UvrD was also found to be necessary to survive DNA damage, but NER or MMR pathways are not completely abolished in UvrD-deficient P. putida. Mfd-deficiency had a moderate impact on surviving DNA damage and did not influence the frequency of mutations occurred in exponentially growing bacteria. However, the absence of Mfd caused approximately a two-fold decline in stationary-phase mutant frequency compared to the P. putida wild-type strain and suppressed the elevated mutant frequency observed in the ΔuvrD strain. Remarkably, the Mfd-deficient strain also formed less UV-induced mutants. These results suggest that in P. putida the Mfd-mediated TCR could be associated with UV- and stationary-phase mutagenesis.

Development of an Automated UV Irradiation Device for Microbial Cell Culture.

Ultraviolet (UV) mutagenesis is a widely used technique to increase bacterial mutation rates in laboratory experiments. UV mutagenesis requires fine regulation of UV dose, because the number of dead cells increases exponentially as the dose increases. Ignoring this hazard can cause extinction of UV-exposed populations. Therefore, an automated system that cooperatively conducts both growth measurement and UV irradiation is needed for efficient UV mutagenesis experiments. To address this task, we constructed an automated UV irradiation device for microbial cell culture. This device can measure cell density and irradiate the bacterial cells with UV light automatically according to the state of cell growth. We demonstrated that this growth feedback control avoided extinction and enabled accumulation of mutations in bacterial genomes at a rapid rate for a long period. Whole-genome sequencing revealed the high accumulation rate, neutrality, and spectrum of UV-induced mutations. These characteristics were all consistent with those obtained by manual UV irradiation. These results indicate that our automated device is useful in accelerating mutation accumulation over a long duration.

Enhanced production of butyric acid by solid-state fermentation of rice polishings by a mutant strain of Clostridium tyrobutyricum

Purpose: To enhance butyric acid production by solid-state fermentation with a hyper-producing mutant of Clostridium tyrobutyricum generated by random mutagenesis.Methods: Wild type C. tyrobutyricum was mutagenized with UV irradiation, nitrous acid, and ethidium bromide to obtain a hyper-producing strain. Various physiochemical parameters were optimized to increase the butyric acid yield.Results: The UV-induced mutant (C.TUV) produced significantly higher concentrations of butyric acid than the wild type parent, nitrous acid-induced, and ethidium bromide-induced strains. C.TUV increased butyric acid production 1.4-fold more than the parent strain. Fermentation with C.TUV with 2.5 g of rice polishings (w/w), a 2 % inoculum volume (v/v), and a 48-h incubation period at 37°C under anaerobic conditions produced 11.63 mg/100 g of butyric acid. The addition of 0.6 % corn steep liquor as a nitrogen source increased the butyric acid concentration to 26.09 mg/100 g.Conclusion: These optimized fermentation parameters on a small scale can be used on a commercial scale to mass-produce butyric acid.Keywords: Butyric acid, Mutant, Clostridium tyrobutyricum, Mutagen, Solid-state fermentation

Sclerotinia sclerotiorum: An Evaluation of Virulence Theories

Oxalic acid production in Sclerotinia sclerotiorum has long been associated with virulence. Research involving UV-induced, genetically undefined mutants that concomitantly lost oxalate accumulation, sclerotial formation, and pathogenicity supported the conclusion that oxalate is an essential pathogenicity determinant of S. sclerotiorum. However, recent investigations showed that genetically defined mutants that lost oxalic acid production but accumulated fumaric acid could cause disease on many plants and substantiated the conclusion that acidic pH, not oxalic acid per se, is the necessary condition for disease development. Critical evaluation of available evidence showed that the UV-induced mutants harbored previously unrecognized confounding genetic defects in saprophytic growth and pH responsiveness, warranting reevaluation of the conclusions about virulence based on the UV-induced mutants. Furthermore, analyses of the evidence suggested a hypothesis for the existence of an unrecognized regulator responsive to acidic pH. Identifying the unknown pH regulator would offer a new avenue for investigating pH sensing/regulation in S. sclerotiorum and novel targets for intervention in disease control strategies.

Ultraviolet radiation-mediated development of cutaneous melanoma: An update

Ultraviolet (UV) light is absorbed by nucleic acids, proteins or other endogenous chromophores, such as porphyrins, flavins and melanin, triggering biological processes in skin cells. Both UV-induced mutations in melanocytes and changes in the immune microenvironment are understood to play a role in the development of cutaneous melanoma. The degree of UV-induced stress and the protection against this stress are influenced by both intracellular and intercellular molecular interactions. The present review summarizes the known major molecular biological changes induced by UV light in the skin that play a role in melanoma initiation and promotion. Nevertheless, cutaneous melanoma is not a homogenous disease, and the interaction of variable environmental exposure and different genetic susceptibility and other host factors lead to the formation of melanomas with different biological behavior and clinical characteristics. This review highlights the challenges in the understanding of how UV radiation contributes to the formation of cutaneous melanoma, and reviews the new results of photobiology and their link to tumor genetics and tumor immunology with potential implications on melanoma prevention and therapeutic strategies. The information presented here is expected to add clarity to ongoing research efforts in this field to aid the development of novel strategies to prevent and treat melanoma.

Statistical optimization of kojic acid production by a UV-induced mutant strain of Aspergillus terreus.

The ability of four Aspergillus strains for biosynthesis of kojic acid was evaluated among which Aspergillus terreus represented the highest level (2.21 g/L) of kojic acid production. Improvement kojic acid production ability of A. terreus by random mutagenesis using different exposure time to ultraviolet light (5–40 min) was then performed to obtain a suitable mutant of kojic acid production (designated as C5-10, 7.63 g/L). Thereafter, design of experiment protocol was employed to find medium components (glucose, yeast extract, KH2PO4 (NH4)2SO4, and pH) influences on kojic acid production by the C5-10 mutant. A 25−1 fractional factorial design augmented to central composite design showed that glucose, yeast extract, and KH2PO4 were the most considerable factors within the tested levels (p < 0.05). The optimum medium composition for the kojic acid production by the C5-10 mutant was found to be glucose, 98.4 g/L; yeast extract, 1.0 g/L; and KH2PO4, 10.3 mM which was theoretically able to produce 120.2 g/L of kojic acid based on the obtained response surface model for medium optimization. Using these medium compositions an experimental maximum Kojic acid production (109.0 ± 10 g/L) was acquired which verified the efficiency of the applied method.

Risk Stratification: Should All Actinic Keratoses in All Patients Be Treated?

Actinic keratoses (AKs) are foci of dysplastic keratinocytes in the epidermis. They are markers of both photodamage and risk of keratinocyte carcinoma (KC) formation. Individual AKs are thought to progress to KC uncommonly, although they arise from the same UV-induced mutations which give rise to KC. Thus, the rationale for treatment of AKs is to reduce the risk of KC formation since AKs are not dangerous in themselves. An estimated $1.68 billion is spent on treatment of AKs in the United States annually. Treatment of a region of skin (e.g., face, scalp, or forearms) to clear AKs is termed field treatment. Though field treatment of AKs and actinic damage has been associated with a reduction in cutaneous squamous cell carcinoma (CSCC), it is unclear to what degree treatment of individual lesions positively impacts health. The presence of AKs has been correlated with formation of KC, particularly CSCC. Field treatment can be accomplished by topical therapy [photodynamic therapy (PDT), topical 5-fluorouracil, ingenol mebutate, imiquimod, and/or sunscreen] or oral systemic therapy including nicotinamide or retinoids (i.e., acitretin, isotretinoin). Field treatments have been associated with a reduction in KC and therefore are likely cost-effective. However, there is a lack of data showing that treatment of individual AK lesions (usually accomplished via cryotherapy) reduces KC. Although cryotherapy is only associated with a 4% sustained clearance rate at 1 year, more than three times the amount of money is spent on cryotherapy as compared to topical therapy in Medicare patients. Field treatment is superior to cryotherapy for reduction of KC. However, it requires high patient compliance and increasingly entails high out-of-pocket expenses for patients. Since field-directed and systemic therapies of AKs reduce KC formation, allocation of resources should be directed toward these modalities. Efforts should be made to improve patient access to field treatments by increased reimbursement for photodynamic therapy (both standard and daylight), reduction of out-of-pocket expenses for topical treatments and retinoids, and increased use of nicotinamide in patients with field actinic damage.

G-101 Human papillomaviruses and carcinogenesis: well-established and novel models

Human papillomaviruses (HPVs) infect the cutaneous or mucosal epithelia. More than 200 HPV types have been isolated so far, and they are classified phylogenetically as genera and species. Persistent infections by the mucosal high-risk (HR) HPV types from genus alpha have been clearly associated with cancer development of the genital and upper respiratory tracts. The products of two early genes, E6 and E7, are the major HR HPV oncoproteins, being essential in all steps of the carcinogenic process. They exert their functions by interacting with a large number of cellular proteins, including the products of tumour suppressor genes, and altering their properties. Biological and epidemiological data indicate that beta HPV types, together with ultraviolet (UV) radiation, promote non-melanoma skin cancer development. However, in contrast to the HR mucosal HPV types, cutaneous beta HPV types appear to be required only at an early stage of carcinogenesis, facilitating the accumulation of UV-induced DNA mutations. Several findings also suggest that these HPV types and other carcinogens may synergize in the induction of malignancies at different anatomical sites.

Small-molecules that bind to the ubiquitin-binding motif of REV1 inhibit REV1 interaction with K164-monoubiquitinated PCNA and suppress DNA damage tolerance

REV1 protein is a mutagenic DNA damage tolerance (DDT) mediator and encodes two ubiquitin-binding motifs (i.e., UBM1 and UBM2) that are essential for the DDT function. REV1 interacts with K164-monoubiquitinated PCNA (UbPCNA) in cells upon DNA-damaging stress. By using AlphaScreen assays to detect inhibition of REV1 and UbPCNA protein interactions along with an NMR-based strategy, we identified small-molecule compounds that inhibit the REV1/UbPCNA interaction and that directly bind to REV1 UBM2. In cells, one of the compound prevented recruitment of REV1 to PCNA foci on chromatin upon cisplatin treatment, delayed removal of UV-induced cyclopyrimidine dimers from nuclei, prevented UV-induced mutation of HPRT gene, and diminished clonogenic survival of cells that were challenged by cyclophosphamide or cisplatin. This study demonstrates the potential utility of a small-molecule REV1 UBM2 inhibitor for preventing DDT.

Sequential decitabine and carboplatin treatment increases the DNA repair protein XPC, increases apoptosis and decreases proliferation in melanoma

BackgroundMelanoma has two key features, an over-representation of UV-induced mutations and resistance to DNA damaging chemotherapy agents. Both of these features may result from dysfunction of the nucleotide excision repair pathway, in particular the DNA damage detection branch, global genome repair (GGR). The key GGR component XPC does not respond to DNA damage in melanoma, the cause of this lack of response has not been investigated. In this study, we investigated the role of methylation in reduced XPC in melanoma.MethodsTo reduce methylation and induce DNA-damage, melanoma cell lines were treated with decitabine and carboplatin, individually and sequentially. Global DNA methylation levels, XPC mRNA and protein expression and methylation of the XPC promoter were examined. Apoptosis, cell proliferation and senescence were also quantified. XPC siRNA was used to determine that the responses seen were reliant on XPC induction.ResultsTreatment with high-dose decitabine resulted in global demethylation, including the the shores of the XPC CpG island and significantly increased XPC mRNA expression. Lower, clinically relevant dose of decitabine also resulted in global demethylation including the CpG island shores and induced XPC in 50% of cell lines. Decitabine followed by DNA-damaging carboplatin treatment led to significantly higher XPC expression in 75% of melanoma cell lines tested. Combined sequential treatment also resulted in a greater apoptotic response in 75% of cell lines compared to carboplatin alone, and significantly slowed cell proliferation, with some melanoma cell lines going into senescence. Inhibiting the increased XPC using siRNA had a small but significant negative effect, indicating that XPC plays a partial role in the response to sequential decitabine and carboplatin.ConclusionsDemethylation using decitabine increased XPC and apoptosis after sequential carboplatin. These results confirm that sequential decitabine and carboplatin requires further investigation as a combination treatment for melanoma.

Photocarcinogenesis and Skin Cancer Prevention Strategies: An Update.

UV radiation is acknowledged as the primary cause of photocarcinogenesis and therefore contributes to the development of skin cancer entities such as squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and melanoma. Typical DNA photoproducts and indirect DNA damage caused by reactive oxygen species are the result of UV radiation. UV-induced DNA damage is repaired by nucleotide excision repair, which consequently counteracts the development of mutations and skin carcinogenesis. Tumour-suppressor genes are inactivated by mutation and growth-promoting pathways are activated leading to disruption of cell-cycle progression. Depending on the skin cancer entity, some genes are more frequently affected than others. In BCC mutations in Patched or Smoothened are common and affect the Sonic hedgehog pathway. In SCC, cell regulator protein p53 (TP53) mutations are prevalent, as well as mutations of the epidermal growth factor receptor (EGFR), cyclin-dependent kinase 2A (CDKN2A), Rat sarcoma (RAS), or the tyrosine kinase Fyn (FYN). UV-induced mutations in TP53 and CDKN2A are frequent in melanoma. UV-induced inflammatory processes also facilitate photocarcinogenesis. Recent studies showed a connection between photocarcinogenesis and citrus consumption, phytochemicals, alcohol consumption, hormone replacement therapy, as well as oral contraceptive use. Preventative measures include adequate use of sun protection and skin cancer screening at regular intervals, as well as the use of chemopreventative agents.

Production of Polyhydroxyalkanoates Copolymers by Recombinant Pseudomonas in Plasmid- and Antibiotic-Free Cultures

Three different polyhydroxyalkanoate (PHA) synthase genes (Ralstonia eutropha H16, Aeromonas sp. TSM81 or Aeromonas hydrophila ATCC7966 phaC) were introduced into the chromosome of two Pseudomonas strains: a native medium-chain-length 3-polyhydroxyalkanoate (PHA_MCL) producer (Pseudomonas sp. LFM046) and a UV-induced mutant strain unable to produce PHA (Pseudomonas sp. LFM461). We reported for the first time the insertion of a chromosomal copy of phaC using the transposon system mini-Tn7. Stable antibiotic marker-free and plasmid-free recombinants were obtained. Subsequently, P(3HB-co-3HA_MCL) was produced by these recombinants using glucose as the sole carbon source, without the need for co-substrates and under antibiotic-free conditions. A recombinant harboring A. hydrophila phaC produced a terpolyester composed of 84.2 mol% of 3-hydroxybutyrate, 6.3 mol% of 3-hydroxyhexanoate, and 9.5 mol% of 3-hydroxydecanoate from only glucose. Hence, we were successful in increasing the industrial potential of Pseudomonas sp. LFM461 strain by producing PHA copolymers containing 3HB and 3HA_MCL using an unrelated carbon source, for the first time in a plasmid- and antibiotic-free bioprocess.

Epidemiology, biology and therapy of Merkel cell carcinoma: conclusions from the EU project IMMOMEC.

Merkel cell carcinoma (MCC) is a highly aggressive, often lethal neuroendocrine cancer. Its carcinogenesis may be either caused by the clonal integration of the Merkel cell polyomavirus into the host genome or by UV-induced mutations. Notably, virally-encoded oncoproteins and UV-induced mutations affect comparable signaling pathways such as RB restriction of cell cycle progression or p53 inactivation. Despite its low incidence, MCC recently received much attention based on its exquisite immunogenicity and the resulting major success of immune modulating therapies. Here, we summarize current knowledge on epidemiology, biology and therapy of MCC as conclusion of the project 'Immune Modulating strategies for treatment of Merkel Cell Carcinoma', which was funded over a 5-year period by the European Commission to investigate innovative immunotherapies for MCC.

Copy number variations in atypical fibroxanthomas and pleomorphic dermal sarcomas.

Atypical fibroxanthomas (AFX) and pleomorphic dermal sarcomas (PDS) are frequent cutaneous sarcomas typically arising on sun-exposed skin in elderly patients. In contrast to AFX, which generally do not recur after complete excision, PDS locally recur in up to 50% and metastasize in up to 20%.We recently detected characteristic UV-induced TP53 mutations as potential driver mutation in almost all PDS investigated as well as activating PIK3CA and RAS gene mutations in around one third of our tumors representing targets for personalized treatments in patients with unresectable or metastasized PDS.In the present study, we identified amplifications and deletions in a small part of the PDS (6 of 27 cases) but not in AFX suggesting that copy number variations (CNV) might not be an initial event in tumor development but rather important during tumor progression. In addition to BRAF, KNSTRN, IDH1 and PDGFRA amplification, CNV analyses revealed deletions in the CDKN2A, KIT and PDGFRA genes. In cases where an appropriate FISH assay was established, the CNV results could be verified by FISH analysis.Amplification of BRAF, KIT or PDGFRA and/or losses of CDKN2A might represent bad prognostic markers, although larger studies are needed to clarify their association with prognosis or progression in PDS.

Mutation accumulation under UV radiation in Escherichia coli

Mutations are induced by not only intrinsic factors such as inherent molecular errors but also by extrinsic mutagenic factors such as UV radiation. Therefore, identifying the mutational properties for both factors is necessary to achieve a comprehensive understanding of evolutionary processes both in nature and in artificial situations. Although there have been extensive studies on intrinsic factors, the mutational profiles of extrinsic factors are poorly understood on a genomic scale. Here, we explored the mutation profiles of UV radiation, a ubiquitous mutagen, in Escherichia coli on the genomic scale. We performed an evolution experiment under periodic UV radiation for 28 days. The accumulation speed of the mutations was found to increase so that it exceeded that of a typical mutator strain with deficient mismatch repair processes. The huge contribution of the extrinsic factors to all mutations consequently increased the risk of the destruction of inherent error correction systems. The spectrum of the UV-induced mutations was broader than that of the spontaneous mutations in the mutator. The broad spectrum and high upper limit of the frequency of occurrence suggested ubiquitous roles for UV radiation in accelerating the evolutionary process.

The effect of phototherapy on progression to tumors in patients with patch and plaque stage of mycosis fungoides

Background: Phototherapy has been a mainstay in the treatment of mycosis fungoides (MF). However, the recent findings of UV-induced p53 mutations in advanced MF suggest that phototherapy may contribute to disease progression.Objective: The objective of this study was to evaluate the effect of phototherapy on the time to tumor progression and overall survival in MF.Materials and methods: Retrospective analysis of patients seen at the University of Pittsburgh Cutaneous Lymphoma Clinic from 1979 to 2016.Results: A total of 345 patients with MF were identified. 258 (74.8%) were diagnosed at stage IA or IB. 43 out of the 258 (16.6%) progressed to tumor stage. Before tumor development, 30 out of the 43 (69.8%) patients received phototherapy, and 13 (30.2%) did not. Patients who received phototherapy had a longer median time to tumor progression than those who did not: 3.5 years (interquartile range = 1.9–5.7) versus 1.2 years (0.2–2.3) (p = .001). Patients who received phototherapy also survived longer: 6.9 years (interquartile range = 4.3–9.5) versus 3.8 years (3.0–4.5) (p = .014).Limitations: Limited information on specific phototherapy start dates, durations, and treatment protocols.Conclusions: The therapeutic effects of phototherapy, with longer times to tumor progression and increased overall survival, appear to outweigh its potential adverse effects.

Carcinogen susceptibility is regulated by genome architecture and predicts cancer mutagenesis.

The development of many sporadic cancers is directly initiated by carcinogen exposure. Carcinogens induce malignancies by creating DNA lesions (i.e., adducts) that can result in mutations if left unrepaired. Despite this knowledge, there has been remarkably little investigation into the regulation of susceptibility to acquire DNA lesions. In this study, we present the first quantitative human genome-wide map of DNA lesions induced by ultraviolet (UV) radiation, the ubiquitous carcinogen in sunlight that causes skin cancer. Remarkably, the pattern of carcinogen susceptibility across the genome of primary cells significantly reflects mutation frequency in malignant melanoma. Surprisingly, DNase-accessible euchromatin is protected from UV, while lamina-associated heterochromatin at the nuclear periphery is vulnerable. Many cancer driver genes have an intrinsic increase in carcinogen susceptibility, including the BRAF oncogene that has the highest mutation frequency in melanoma. These findings provide a genome-wide snapshot of DNA injuries at the earliest stage of carcinogenesis. Furthermore, they identify carcinogen susceptibility as an origin of genome instability that is regulated by nuclear architecture and mirrors mutagenesis in cancer.

A review of BF-200 ALA for the photodynamic treatment of mild-to-moderate actinic keratosis.

BF-200 ALA is a combination of a nanoscale-lipid vesicle formulation and the prodrug 5-aminolevulinic acid (5-ALA). The nanoemulsion stabilizes the prodrug and enhances its penetration through the stratum corneum. It has shown excellent therapeutic results in both lesion and field-directed photodynamic therapy of actinic keratosis (AK). AK is an early form of epidermal neoplasia and a precursor of invasive cutaneous squamous cell carcinoma. It is characterized by the combination of visible neoplastic lesions and surrounding tissue also harboring tumorigenic UV-induced mutations: a concept called field cancerization. A selective, field-directed treatment is ideal to meet the requirements of field change. Here, we review the clinical data on BF-200 ALA for AK along with a summary of molecular mechanisms and future perspectives.

Stimulation of RNA Polymerase II ubiquitination and degradation by yeast mRNA 3′-end processing factors is a conserved DNA damage response in eukaryotes

The quality and retrieval of genetic information is imperative to the survival and reproduction of all living cells. Ultraviolet (UV) light induces lesions that obstruct DNA access during transcription, replication, and repair. Failure to remove UV-induced lesions can abrogate gene expression and cell division, resulting in permanent DNA mutations. To defend against UV damage, cells utilize transcription-coupled nucleotide excision repair (TC-NER) to quickly target lesions within active genes. In cases of long-term genotoxic stress, a slower alternative pathway promotes degradation of RNA Polymerase II (Pol II) to allow for global genomic nucleotide excision repair (GG-NER). The crosstalk between TC-NER and GG-NER pathways and the extent of their coordination with other nuclear events has remained elusive. We aimed to identify functional links between the DNA damage response (DDR) and the mRNA 3′-end processing complex. Our labs have previously shown that UV-induced inhibition of mRNA processing is a conserved DDR between yeast and mammalian cells. Here we have identified mutations in the yeast mRNA 3′-end processing cleavage factor IA (CFIA) and cleavage and polyadenylation factor (CPF) that confer sensitivity to UV-type DNA damage. In the absence of TC-NER, CFIA and CPF mutants show reduced UV tolerance and an increased frequency of UV-induced genomic mutations, consistent with a role for RNA processing factors in an alternative DNA repair pathway. CFIA and CPF mutants impaired the ubiquitination and degradation of Pol II following DNA damage, but the co-transcriptional recruitment of Pol II degradation factors Elc1 and Def1 was undiminished. Overall these data are consistent with yeast 3′-end processing factors contributing to the removal of Pol II stalled at UV-type DNA lesions, a functional interaction that is conserved between homologous factors in yeast and human cells.

Strain improvement of Lentzea sp. 7887 for higher yield per unit volume on hydroxylation of cyclosporine derivative FR901459.

A Gram-positive bacterium Lentzea sp. 7887 hydroxylates a cyclosporine derivative FR901459 into AS1837812 (9-hydroxide), which is an important intermediate of candidate drugs that target the hepatitis C virus. We screened a UV-induced mutant, named M-1, which showed about 1.2-fold higher conversion yields, 2-fold higher substrate concentrations (3.69 mM), and 2.5-fold higher yield per unit volume than the wild-type strain.