Pyrimidine Dimers In DNA Research Articles

Data Processing for Predicting DNA Damaging Properties of Complex UV Sources.

A growing number of experimental evidence emphasizes that photobiological phenomena are not always the sum of the effect of individual wavelengths present in the emission spectrum of light sources. Unfortunately, tools are missing to identify such non-additive effects and predict effects of various exposure conditions. In the present work, we addressed these points for the formation of pyrimidine dimers in DNA upon co-exposure to UVC, UVB and UVA radiation. We first applied a combination index approach to determine whether mixtures of theses UV ranges exhibited additive, inhibitory or synergistic effects on the formation of cyclobutane pyrimidine dimers, (6-4) photoproducts and Dewar valence isomers. A predictive approach based on an experimental design strategy was then used to quantify the contribution of each wavelength range to the formation of DNA photoproducts. The obtained models allowed us to accurately predict the level of pyrimidine dimers in DNA irradiated under different conditions. The data were found to be more accurate than those obtained with the simple additive approach underlying the use of action spectra. Experimental design thus appears as an attractive concept that could be widely applied in photobiology even for cellular experiments.

Identification of a Noxo1 inhibitor by addition of a polyethylene glycol chain

Reactive oxygen species (ROS) are a heterogeneous group of highly reactive ions and molecules derived from molecular oxygen (O2) which can cause DNA damage and lead to skin cancer. NADPH oxidase 1 (Nox1) is a major producer of ROS in the skin upon exposure to ultraviolet light. Functionally, Nox1 forms a holoenzyme complex that generates two superoxide molecules and reduces NADPH. The signaling activation occurs when the organizer subunit Noxo1 translocates to the plasma membrane bringing a cytochrome p450, through interaction with Cyba. We propose to design inhibitors that prevent Cyba-Noxo1 binding as a topical application to reduce UV-generated ROS in human skin cells. Design started from an apocynin backbone structure to generate a small molecule to serve as an anchor point. The initial compound was then modified by addition of a polyethylene glycol linked biotin. Both inhibitors were found to be non-toxic in human keratinocyte cells. Further in vitro experiments using isothermal calorimetric binding quantification showed the modified biotinylated compound bound Noxo1 peptide with a KD of 2 nM. Both using isothermal calorimetric binding and MALDI (TOF) MS showed that binding of a Cyba peptide to Noxo1 was blocked. In vivo experiments were performed using donated skin explants with topical application of the two inhibitors. Experiments show that ultraviolet light exposure of with the lead compound was able to reduce the amount of cyclobutene pyrimidine dimers in DNA, a molecule known to lead to carcinogenesis. Further synthesis showed that the polyethylene glycol but not the biotin was essential for inhibition.

An Estuarine Cyanophage S-CREM1 Encodes Three Distinct Antitoxin Genes and a Large Number of Non-Coding RNA Genes.

Cyanophages play important roles in regulating the population dynamics, community structure, metabolism, and evolution of cyanobacteria in aquatic ecosystems. Here, we report the genomic analysis of an estuarine cyanophage, S-CREM1, which represents a new genus of T4-like cyanomyovirus and exhibits new genetic characteristics. S-CREM1 is a lytic phage which infects estuarine Synechococcus sp. CB0101. In contrast to many cyanomyoviruses that usually have a broad host range, S-CREM1 only infected the original host strain. In addition to cyanophage-featured auxiliary metabolic genes (AMGs), S-CREM1 also contains unique AMGs, including three antitoxin genes, a MoxR family ATPase gene, and a pyrimidine dimer DNA glycosylase gene. The finding of three antitoxin genes in S-CREM1 implies a possible phage control of host cells during infection. One small RNA (sRNA) gene and three cis-regulatory RNA genes in the S-CREM1 genome suggest potential molecular regulations of host metabolism by the phage. In addition, S-CREM1 contains a large number of tRNA genes which may reflect a genomic adaption to the nutrient-rich environment. Our study suggests that we are still far from understanding the viral diversity in nature, and the complicated virus-host interactions remain to be discovered. The isolation and characterization of S-CREM1 further our understanding of the gene diversity of cyanophages and phage-host interactions in the estuarine environment.

The ubiquitin-binding domain of DNA polymerase η directly binds to DNA clamp PCNA and regulates translesion DNA synthesis

DNA polymerase eta (Polη) is a unique translesion DNA synthesis (TLS) enzyme required for the error-free bypass of ultraviolet ray (UV)-induced cyclobutane pyrimidine dimers in DNA. Therefore, its deficiency confers cellular sensitivity to UV radiation and an increased rate of UV-induced mutagenesis. Polη possesses a ubiquitin-binding zinc finger (ubz) domain and a PCNA-interacting-protein (pip) motif in the carboxy-terminal region. The role of the Polη pip motif in PCNA interaction required for DNA polymerase recruitment to the stalled replication fork has been demonstrated in earlier studies; however, the function of the ubz domain remains divisive. As per the current notion, the ubz domain of Polη binds to the ubiquitin moiety of the ubiquitinated PCNA, but such interaction is found to be nonessential for Polη's function. In this study, through amino acid sequence alignments, we identify three classes of Polη among different species based on the presence or absence of pip motif or ubz domain and using comprehensive mutational analyses, we show that the ubz domain of Polη, which intrinsically lacks the pip motif directly binds to the interdomain connecting loop (IDCL) of PCNA and regulates Polη's TLS activity. We further propose two distinct modes of PCNA interaction mediated either by pip motif or ubz domain in various Polη homologs. When the pip motif or ubz domain of a given Polη binds to the IDCL of PCNA, such interaction becomes essential, whereas the binding of ubz domain to PCNA through ubiquitin is dispensable for Polη's function.

Solar Simulated Light Induces Cutaneous Squamous Cell Carcinoma in Inbred Mice: A Clinically Relevant Model to Investigate T-Cell Responses

More than one million cutaneous squamous cell carcinomas (cSCCs) are diagnosed annually in the United States (Rogers et al., 2015). The main causal factor of cSCC is UV exposure from sunlight, which is made of 95% UVA and 5% UVB at the Earth’s surface. UVA and UVB cause cyclobutane pyrimidine dimers in DNA and oxidative damage through the formation of ROS. The ratio of cyclobutane pyrimidine dimers to oxidative DNA damage products is ∼800 times greater in UVB than in UVA (Zhang et al., 1997). In addition, UVA and UVB have distinct effects on keratinocyte signaling pathways, including apoptosis (Bito et al., 2010; Syed et al., 2012).

Appropriate Technologies to Accompany Sunscreens in the Battle Against Ultraviolet, Superoxide, and Singlet Oxygen.

The interaction of ultraviolet radiation with biological matter results in direct damage such as pyrimidine dimers in DNA. It also results in indirect damage provoked by the production of reactive oxygen species (ROS) catalyzed by photosensitizers. Photosensitizers can be endogenous (e.g., tryptophan) or exogenous (e.g., TiO2 and other photostable UVA sunscreens). Direct damage triggers an inflammatory response and the oxidative and proteolytic bursts that characterize its onset. The inflammatory reaction multiplies the effects of one single photon. Indirect damage, such as the peroxidative cascade in membrane lipids, can extend to thousands of molecular modifications per absorbed photon. Sunscreens should therefore be formulated in the presence of appropriate antioxidants. Superoxide and singlet oxygen are the main ROS that need to be tackled: this review describes some of the molecular, biochemical, cellular, and clinical consequences of exposure to UV radiation as well as some results associated with scavengers and quenchers of superoxide and singlet oxygen, as well as with inhibitors of singlet oxygen production.

Pyrimidine (6‐4) Pyrimidone Photoproducts in UVA‐Irradiated DNA: Photosensitization or Photoisomerization?

AbstractFormation of pyrimidine dimers in DNA is a major initiating event in the induction of skin cancer. Model experiments suggest that, upon absorption of UVA, one type of dimers induced by UVB, the pyrimidine (6‐4) pyrimidone photoproducts, photosensitizes the formation of mutagenic cyclobutane pyrimidine dimers by triplet ‐triplet energy transfer (TTET). We investigated whether this photoreaction actually took place when 64PP were located within a DNA duplex rather than added as external sensitizers like in available data. Our results show that this process is not detectable in DNA and double‐stranded oligonucleotides exposed to a combination of UVB and UVA. TTET could only be observed, as a very minor photoreaction, in a short single‐stranded oligonucleotide bearing a 64PP. It may be concluded that 64PP‐mediated TTET does not significantly contribute to UV‐induced DNA damage. In contrast, the photoisomerization of 64PP into their Dewar valence isomers is very efficient.

Corrigendum: Photochemical Formation of Cyclobutane Pyrimidine Dimers in DNA through Electron Transfer from a Flanking Base.

Electron transfer (ET) to a pyrimidine base from external moieties is a common step involved in the quenching or repair of the cyclobutane pyrimidine dimer (CPD). In contrast, we present a pathway that is initiated by an ET from a flanking guanine base to a pyrimidine base, leading to the formation of a CPD. We studied a T5mCG sequence with a methylated cytosine and our results demonstrate that the pathway involves the formation of an exciplex and intersystem crossings. This pathway also provides an explanation for why the mutational hot spots are correlated with the methylated CpG sequences, which has been a significant issue in cancer research.

Role of charge transfer states into the formation of cyclobutane pyrimidine dimers in DNA

Photoinduced charge transfer between neighboring bases plays an important role in DNA. One of its important effects is shown in its ability to affect the photochemical yields of the formation of cyclobutane pyrimidine dimer (CPD) products between adjacent pyrimidine bases. In this work we examine how the energies of charge transfer states depend on the sequences of oligonucleotides using a hybrid quantum and molecular mechanics (QM/MM) methodology combined with the algebraic diagrammatic construction through a second order electronic structure method for excited states. Specifically, we examine 10 sequences with guanine being on the 5' or 3' position of two pyrimidine bases. The results show that the energies of charge transfer states are affected by the nature of the donor acceptor pair, by the distance between them, and by other electrostatic effects created by the surrounding environment.

Correction: Role of charge transfer states into the formation of cyclobutane pyrimidine dimers in DNA.

Correction: Role of charge transfer states into the formation of cyclobutane pyrimidine dimers in DNA.

Cover Feature: Photochemical Formation of Cyclobutane Pyrimidine Dimers in DNA through Electron Transfer from a Flanking Base (ChemPhysChem 13/2018)

Cover Feature: Photochemical Formation of Cyclobutane Pyrimidine Dimers in DNA through Electron Transfer from a Flanking Base (ChemPhysChem 13/2018)

Photochemical Formation of Cyclobutane Pyrimidine Dimers in DNA through Electron Transfer from a Flanking Base.

Electron transfer (ET) to a pyrimidine base from external moieties is a common step involved in the quenching or repair of the cyclobutane pyrimidine dimer (CPD). In contrast, we present a pathway that is initiated by an ET from a flanking guanine base to a pyrimidine base, leading to the formation of a CPD. We studied a T5mCG sequence with a methylated cytosine and our results demonstrate that the pathway involves the formation of an exciplex and intersystem crossings. This pathway also provides an explanation for why the mutational hot spots are correlated with the methylated CpG sequences, which has been a significant issue in cancer research.

Visual detection of cyclobutane pyrimidine dimer DNA damage lesions by Hg2+ and carbon dots

Cyclobutane pyrimidine dimmers (CPDs) and 6–4-[pyrimidine-2′-one] pyrimidine (6-4 PP) are major UV induced DNA damage lesions formed from solar radiation and other sources. CPD lesions are presumably mutagenic and carcinogenic that inhibit polymerases and interfere in DNA replication. An easy and cost effective way for visual detection of these lesions by using fluorescence based method is shown here. Artificial UVA and UVB lights were used for the generation of CPD and 6-4 PPs in selected DNA samples. Binding of Hg2+ ions with DNA before and after induction of CPD and 6-4 PP lesions was evaluated in the presence of highly fluorescent blue emitting carbon dots (CDs). Induction of CPD and 6-4 PPs in DNA causes distortion of DNA structure which hinders the binding of Hg2+ ions to DNA nucleobases. Quenching of fluorescence intensity of CDs by unbound Hg2+ ions was found to be proportional to the amount of CPD and 6-4 PP lesions induced by UV irradiation of DNA samples that offer a biosensing platform for the sensitive detection of CPD lesions in DNA. The fluorescent quenching was visually detectable using hand held UV light without the intervention of any equipment.

Oxidatively Generated Lesions as Internal Photosensitizers for Pyrimidine Dimerization in DNA.

In this work, the attention is focused on UVA-photosensitized reactions triggered by a DNA chromophore-containing lesion, namely 5-formyluracil. This is a major oxidatively generated lesion that exhibits an enhanced light absorption in the UVB-UVA region. The mechanistic study combining photochemical and photobiological techniques shows that irradiation of 5-formyluracil leads to a triplet excited state capable of sensitizing formation of cyclobutane pyrimidine dimers in DNA via a triplet-triplet energy transfer. This demonstrates for the first time that oxidatively generated DNA damage can behave as an intrinsic sensitizer and result in an important extension of the active fraction of the solar spectrum with photocarcinogenic potential. Overall, this raises the question of an aggravated photomutagenicity of the 5-formyluracil lesion.

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.

Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli.

Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs) becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB), II and V to make mutations during repair. Pol IV is implied to compete with and replace high-fidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR); damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS). First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG) in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cis-acting switch to MBR occurs independently of DNA breakage, caused by ROS oxidation of DNA potentially regulated by ROS regulators.

Insight in DNA Repair of UV-induced Pyrimidine Dimers by Chromatographic Methods.

UV-induced formation of pyrimidine dimers in DNA is a major deleterious event in both eukaryotic and prokaryotic cells. Accumulation of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts can lead to cell death or be at the origin of mutations. In skin, UV induction of DNA damage is a major initiating event in tumorigenesis. To counteract these deleterious effects, all cell types possess DNA repair machinery, such as nucleotide excision repair and, in some cell types, direct reversion. Different analytical approaches were used to assess the efficiency of repair and decipher the enzymatic mechanisms. We presently review the information provided by chromatographic methods, which are complementary to biochemical assays, such as immunological detection and electrophoresis-based techniques. Chromatographic assays are interesting in their ability to provide quantitative data on a wide range of damage and are also valuable tools for the identification of repair intermediates.

A history of the DNA repair and mutagenesis field: I. The discovery of enzymatic photoreactivation.

Photoreactivation, an enzyme-catalyzed reaction during which two covalently linked pyrimidine dimers in DNA are monomerized and restored to their native conformation was the first DNA repair mechanism to be discovered, an event that transpired in the late 1940's through the efforts of the American biologist Albert Kelner while at the Cold Spring Harbor Laboratories in upstate New York. The phenomenon was Independently observed by Renato Dulbecco shortly thereafter, then a post-doctoral fellow in Salvador Luria's Laboratory in Bloomington Indiana. However, Luria and Dulbecco yielded priority to Kelner's discovery.

The significance of the Dewar valence photoisomer as a UV radiation‐induced DNA photoproduct in marine microbial communities

Induction of pyrimidine dimers in DNA by solar UV radiation has drastic effects on microorganisms. To better define the nature of these DNA photoproducts in marine bacterioplankton and eukaryotes, a study was performed during a cruise along a latitudinal transect in the Pacific Ocean. The frequency of all possible cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts (64PPs) and their related Dewar valence isomers (DEWs) was determined by high-performance liquid chromatography-mass spectrometry. Studied samples were bacterioplankton and eukaryotic fractions isolated from sea water either collected before sunrise or exposed to ambient sunlight from sunrise to sunset. Isolated DNA dosimeters were also exposed to daily sunlight for comparison purposes. A first major result was the observation in all samples of large amounts of DEWs, a class of photoproducts rarely considered outside photochemical studies. Evidence was obtained for a major role of UVA in the formation of these photoisomerization products of 64PPs. Considerations on the ratio between the different classes of photoproducts in basal and induced DNA damage suggests that photoenzymatic repair (PER) is an important DNA repair mechanism used by marine microorganisms occupying surface seawater in the open ocean. This result emphasizes the biological role of DEWs which are very poor substrate for PER.

Polymorphisms in the p27 kip-1 and prohibitin genes denote novel genes associated with melanoma risk in Brazil, a high ultraviolet index region

Ultraviolet (UV) radiation is a major environmental risk factor to the development of cutaneous melanoma as it induces pyrimidine dimers in DNA. Genes that exert their function by arresting the cell cycle are critical to avoid carcinogenic mutations, allowing the processing of DNA repair systems. This study was carried out to evaluate the role of polymorphisms in cell cycle genes such as TP53, p27, CDKN2A, prohibitin, and GADD153 in melanoma risk as well as their influence on known risk factors in a high UV index region. A hospital-based case-control study was carried out in Brazil to evaluate the contribution of polymorphisms in cell cycle genes toward melanoma risk. The study comprised 202 melanoma patients and 210 controls. The polymorphisms analyzed were TP53 Arg72Pro, p27 Val109Gly, GADD153 Phe10Phe (rs697221), CDKN2A 3'UTR C540G, and prohibitin 3'UTR C1703T. As regards, p27 Val109Gly, both heterozygous and homozygous Gly genotypes were shown to be protective genotypes on calculating both crude and adjusted odds ratios (ORs) for age, sex, and educational level [OR 0.37; 95% confidence interval (CI) 0.16-0.87; P<0.05]. Similarly, the prohibitin TT genotype increased melanoma risk in the crude and adjusted analyses (OR 2.40; 95% CI 1.10-5.26; P<0.05). The p27 Gly protective genotype decreased the risk for melanoma in a stratified analysis of the known risk factors such as hair and eye color, sunburns, pigmented lesions, and European ancestry. The prohibitin TT genotype increased the risk of melanoma by such host factors. Our results showed for the first time that polymorphisms in p27 Val109Gly and in prohibitin 3'UTR C1703T genotypes modulate the risk to melanoma in a high UV index region.