Cyclobutane Pyrimidine Dimers In DNA Research Articles

Mapping the recognition pathway of cyclobutane pyrimidine dimer in DNA by Rad4/XPC.

UV radiation-induced DNA damages have adverse effects on genome integrity and cellular function. The most prevalent UV-induced DNA lesion is the cyclobutane pyrimidine dimer (CPD), which can cause skin disorders and cancers in humans. Rad4/XPC is a damage sensing protein that recognizes and repairs CPD lesions with high fidelity. However, the molecular mechanism of how Rad4/XPC interrogates CPD lesions remains elusive. Emerging viewpoints indicate that the association of Rad4/XPC with DNA, the insertion of a lesion-sensing β-hairpin of Rad4/XPC into the lesion siteand the flipping of CPD's partner bases (5'-dA and 3'-dA) are essential for damage recognition. Characterizing these slow events is challenging due to their infrequent occurrence on molecular time scales. Herein, we have used enhanced sampling and molecular dynamics simulations to investigate the mechanism and energetics of lesion recognition by Rad4/XPC, considering multiple plausible pathways between the crystal structure of the Rad4-DNA complex and nine intermediate states. Our results shed light on the most likely sequence of events, their potential coupling and energetics. Upon association, Rad4 and DNA form an encounter complex in which CPD and its partner bases remain in the duplex and the BHD3 β-hairpin is yet to be inserted into the lesion site. Subsequently, sequential base flipping occurs, with the flipping of the 5'-dA base preceding that of the 3'-dA base, followed by the insertion of the BHD3 β-hairpin into the lesion site. The results presented here have significant implications for understanding the molecular basis of UV-related skin disorders and cancers and for paving the way for novel therapeutic strategies.

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).

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.

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.

Identification and Purification of the CPD Photolyase in Vibrio parahaemolyticus RIMD2210633.

Photoreactivation is an error-free mechanism of DNA repair, utilized by prokaryotes and most eukaryotes and is catalyzed by specific enzymes called DNA photolyases. Photoreactivation has been reported in Vibrio parahaemolyticus WP28; however, information on photolyases in V. parahaemolyticus (V.p) strains has not been reported. This study examined the photoreactivation in V.p RIMD2210633. The photolyase responsible for repairing cyclobutane pyrimidine dimer (CPD) in DNA was identified, and the corresponding gene was determined as VPA1471. The protein was overexpressed in Escherichia coli and was purified for functional assessment invitro. The mRNA level and protein expression level of this gene increased after ultraviolet A (UVA) illumination following ultraviolet C (UVC) irradiation. In vitro experiments confirmed that the protein encoded by VPA1471 could reduce the quantity of CPD in DNA. We designated the corresponding gene and protein of VPA1471 phr and Phr, respectively, although the function of two other photolyase/cryptochrome family members, VPA0203 and VPA0204, remains unclear. UV (ultraviolet) irradiation experiments suggest that these two genes possess some photorepairing ability. Therefore, we hypothesize that VPA0203 and VPA0204 encode (6-4) photolyase in V.parahaemolyticus RIMD2210633.

The relationship between UV-irradiance, photoprotective compounds and DNA damage in two intertidal invertebrates with contrasting mobility characteristics

The photoprotective role of mycosporine-like amino acids (MAA) against the generation of DNA cyclobutane pyrimidine dimers (CPD) was studied in the sessile intertidal anemone Actinia tenebrosa and the mobile intertidal gastropod Diloma aethiops through 27months at a mid-latitude New Zealand location. MAA were sequestered by A. tenebrosa and D. aethiops from their diet, although maximum total MAA levels in both species were not correlated with seasonal variation in maximum ambient UV-B levels recorded at the collection site. Temporal changes in total MAA in A. tenebrosa showed a six months lag-time in their concentration regarding to the environmental UV-B levels. This lag period corresponded to an observed increase in CPD production from spring to summer; suggesting that MAA do not completely protect the anemone from UV-B during summer. For D. aethiops, total MAA concentrations did not change significantly during the study, although qualitative changes in MAA were apparent. A month lag-time in MAA concentration in D. aethiops and possibly the physical barrier that the shell confers to the animal, can explain reduced CPD levels in comparative terms with A. tenebrosa. Although MAA are used by invertebrates for photoprotection, contrasting mobility characteristics and the presence of physical adaptations can confer them important protection levels during temporal changes of UV-B at mid-latitude places of the Southern Hemisphere.

Global Contributions to the Understanding of DNA Repair and Skin Cancer

Living organisms exposed to sunlight have developed mechanisms to repair the damage that UVR induces in their DNA. These DNA repair systems are present in most life forms including bacteria, yeast, rodents, and mammals. Three rare human genetic diseases have defects in the DNA repair system called nucleotide excision repair (NER). Patients with xeroderma pigmentosum (XP) have defective NER and a more than 10,000-fold increased frequency of skin cancer, whereas patients with Cockayne syndrome (CS) or with trichothiodystrophy (TTD) have NER defects without increase in cancer. These are strikingly different clinical phenotypes (Figure 1). The dramatic differences in their manifestations reflect the complexity of DNA repair and the broad scope and central role of DNA repair on preservation of diverse biological functions. This article will review the major milestones in our understanding of DNA repair and skin cancer (Table 1). These include recognition of the clinical diseases, understanding the types of damage sunlight causes in the DNA, recognition of DNA repair in different organisms, discovery of defective DNA repair in humans, cloning of multiple human DNA repair genes, and identification of UV-type mutations in skin cancer.

Cyclobutane Pyrimidine Dimer Density as a Predictive Biomarker of the Biological Effects of Ultraviolet Radiation in Normal Human Fibroblast

This study compared biological responses of normal human fibroblasts (NHF1) to three sources of ultraviolet radiation (UVR), emitting UVC wavelengths, UVB wavelengths, or a combination of UVA and UVB (solar simulator; emission spectrum, 94.3% UVA and 5.7% UVB). The endpoints measured were cytotoxicity, intra-S checkpoint activation, inhibition of DNA replication and mutagenicity. Results show that the magnitude of each response to the indicated radiation sources was best predicted by the density of DNA cyclobutane pyrimidine dimers (CPD). The density of 6-4 pyrimidine-pyrimidone photoproducts was highest in DNA from UVC-irradiated cells (14% of CPD) as compared to those exposed to UVB (11%) or UVA-UVB (7%). The solar simulator source, under the experimental conditions described here, did not induce the formation of 8-oxo-7,8-dihydroguanine in NHF1 above background levels. Taken together, these results suggest that CPD play a dominant role in DNA damage responses and highlight the importance of using endogenous biomarkers to compare and report biological effects induced by different sources of UVR.

Cyclobutane pyrimidine dimer (CPD) photolyase repairs ultraviolet‐B‐induced CPDs in rice chloroplast and mitochondrial DNA

Plants use sunlight as energy for photosynthesis; however, plant DNA is exposed to the harmful effects of ultraviolet-B (UV-B) radiation (280-320 nm) in the process. UV-B radiation damages nuclear, chloroplast and mitochondrial DNA by the formation of cyclobutane pyrimidine dimers (CPDs), which are the primary UV-B-induced DNA lesions, and are a principal cause of UV-B-induced growth inhibition in plants. Repair of CPDs is therefore essential for plant survival while exposed to UV-B-containing sunlight. Nuclear repair of the UV-B-induced CPDs involves the photoreversal of CPDs, photoreactivation, which is mediated by CPD photolyase that monomerizes the CPDs in DNA by using the energy of near-UV and visible light (300-500 nm). To date, the CPD repair processes in plant chloroplasts and mitochondria remain poorly understood. Here, we report the photoreactivation of CPDs in chloroplast and mitochondrial DNA in rice. Biochemical and subcellular localization analyses using rice strains with different levels of CPD photolyase activity and transgenic rice strains showed that full-length CPD photolyase is encoded by a single gene, not a splice variant, and is expressed and targeted not only to nuclei but also to chloroplasts and mitochondria. The results indicate that rice may have evolved a CPD photolyase that functions in chloroplasts, mitochondria and nuclei, and that contains DNA to protect cells from the harmful effects of UV-B radiation.

Quantification of Cyclobutane Pyrimidine Dimers Induced by UVB Radiation in Conidia of the Fungi Aspergillus fumigatus, Aspergillus nidulans, Metarhizium acridum and Metarhizium robertsii

Conidia are responsible for reproduction, dispersal, environmental persistence and host infection of many fungal species. One of the main environmental factors that can kill and/or damage conidia is solar UV radiation. Cyclobutane pyrimidine dimers (CPD) are the major DNA photoproducts induced by UVB. We examined the conidial germination kinetics and the occurrence of CPD in DNA of conidia exposed to different doses of UVB radiation. Conidia of Aspergillus fumigatus, Aspergillus nidulans and Metarhizium acridum were exposed to UVB doses of 0.9, 1.8, 3.6 and 5.4 kJ m(-2). CPD were quantified using T4 endonuclease V and alkaline agarose gel electrophoresis. Most of the doses were sublethal for all three species. Exposures to UVB delayed conidial germination and the delays were directly related both to UVB doses and CPD frequencies. The frequencies of dimers also were linear and directly proportional to the UVB doses, but the CPD yields differed among species. We also evaluated the impact of conidial pigmentation on germination and CPD induction on Metarhizium robertsii. The frequency of dimers in an albino mutant was approximately 10 times higher than of its green wild-type parent strain after exposure to a sublethal dose (1.8 kJ m(-2)) of UVB radiation.

Quantification of UV-induced cyclobutane pyrimidine dimers using an oligonucleotide chip assay

A lesion-specific enzyme-induced DNA strand break assay was developed for an oligonucleotide chip for the determination of UVB-induced cyclobutane pyrimidine dimers (CPDs). A 20-mer of fluorophore-labeled and biotinylated oligonucleotide was immobilized on the chip. CPDs in DNA on the chip were formed by UVB irradiation (312 nm). T4 endonuclease V (T4N5) was used to excise the CPD site as T4N5 sensitively and specifically detects CPDs. The fluorophore-labeled DNA fragments were detected by a laser-induced fluorescence (LIF) detection system. The number of CPDs induced by UVB was determined based on a mathematical equation obtained from a predetermined calibration curve. The yield of UVB-induced CPDs was 1.73 CPDs per megabase per (kJ/m(2)). The reliability of this value was proved by its similarity to reference values obtained from gel electrophoresis. The developed assay has strong potential to quantify most kinds of UV-induced DNA lesions.

UVA-induced cyclobutane pyrimidine dimers in DNA: a direct photochemical mechanism?

The carcinogenic action of UVA radiation is commonly attributed to DNA oxidation mediated by endogenous photosensitisers. Yet, it was recently shown that cyclobutane pyrimidine dimers (CPD), well known for their involvement in UVB genotoxicity, are produced in larger yield than oxidative lesions in UVA-irradiated cells and skin. In the present work, we gathered mechanistic information on this photoreaction by comparing formation of all possible bipyrimidine photoproducts upon UVA irradiation of cells, purified genomic DNA and dA(20):dT(20) oligonucleotide duplex. We observed that the distribution of photoproducts, characterized by the sole formation of CPD and the absence of (6-4) photoproducts was similar in the three types of samples. The CPD involving two thymines represented 90% of the amount of photoproducts. Moreover, the yields of formation of the DNA lesions were similar in cells and isolated DNA. In addition, the effect of the wavelength of the incident photons was found to be the same in isolated DNA and cells. This set of data shows that UVA-induced cyclobutane pyrimidine dimers are formed via a direct photochemical mechanism, without mediation of a cellular photosensitiser. This is possible because the double-stranded structure increases the capacity of DNA bases to absorb UVA photons, as evidenced in the case of the oligomer dA(20):dT(20). These results emphasize the need to consider UVA in the carcinogenic effects of sunlight. An efficient photoprotection is needed that can only be complete by completely blocking incident photons, rather than by systemic approaches such as antioxidant supplementation.

DNA photolyases of Chrysodeixis chalcites nucleopolyhedrovirus are targeted to the nucleus and interact with chromosomes and mitotic spindle structures

Cyclobutane pyrimidine dimer (CPD) photolyases convert UV-induced CPDs in DNA into monomers using visible light as the energy source. Two phr genes encoding class II CPD photolyases PHR1 and PHR2 have been identified in Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV). Transient expression assays in insect cells showed that PHR1-EGFP fusion protein was localized in the nucleus. Early after transfection, PHR2-EGFP was distributed over the cytoplasm and nucleus but, over time, it became localized predominantly in the nucleus. Immunofluorescence analysis with anti-PHR2 antiserum showed that, early after transfection, non-fused PHR2 was already present mainly in the nucleus, suggesting that the fusion of PHR2 to EGFP hindered its nuclear import. Both PHR-EGFP fusion proteins strongly colocalized with chromosomes and spindle, aster and midbody structures during host-cell mitosis. When PHR2-EGFP-transfected cells were superinfected with Autographa californica multiple-nucleocapsid NPV (AcMNPV), the protein colocalized with virogenic stroma, the replication factories of baculovirus DNA. The collective data support the supposition that the PHR2 protein plays a role in baculovirus DNA repair.

UVA Generates Pyrimidine Dimers in DNA Directly

There is increasing evidence that UVA radiation, which makes up ∼95% of the solar UV light reaching the Earth's surface and is also commonly used for cosmetic purposes, is genotoxic. However, in contrast to UVC and UVB, the mechanisms by which UVA produces various DNA lesions are still unclear. In addition, the relative amounts of various types of UVA lesions and their mutagenic significance are also a subject of debate. Here, we exploit atomic force microscopy (AFM) imaging of individual DNA molecules, alone and in complexes with a suite of DNA repair enzymes and antibodies, to directly quantify UVA damage and reexamine its basic mechanisms at a single-molecule level. By combining the activity of endonuclease IV and T4 endonuclease V on highly purified and UVA-irradiated pUC18 plasmids, we show by direct AFM imaging that UVA produces a significant amount of abasic sites and cyclobutane pyrimidine dimers (CPDs). However, we find that only ∼60% of the T4 endonuclease V-sensitive sites, which are commonly counted as CPDs, are true CPDs; the other 40% are abasic sites. Most importantly, our results obtained by AFM imaging of highly purified native and synthetic DNA using T4 endonuclease V, photolyase, and anti-CPD antibodies strongly suggest that CPDs are produced by UVA directly. Thus, our observations contradict the predominant view that as-yet-unidentified photosensitizers are required to transfer the energy of UVA to DNA to produce CPDs. Our results may help to resolve the long-standing controversy about the origin of UVA-produced CPDs in DNA.

Active Site of Escherichia coli DNA Photolyase: Asn378 Is Crucial both for Stabilizing the Neutral Flavin Radical Cofactor and for DNA Repair

Escherichia coli DNA photolyase repairs cyclobutane pyrimidine dimer (CPD) in UV-damaged DNA through a photoinduced electron transfer mechanism. The catalytic activity of the enzyme requires fully reduced FAD (FADH (-)). After purification in vitro, the cofactor FADH (-) in photolyase is oxidized into the neutral radical form FADH (*) under aerobic conditions and the enzyme loses its repair function. We have constructed a mutant photolyase in which asparagine 378 (N378) is replaced with serine (S). In comparison with wild-type photolyase, we found N378S mutant photolyase containing oxidized FAD (FAD ox) but not FADH (*) after routine purification procedures, but evidence shows that the mutant protein contains FADH (-) in vivo as the wild type. Although N378S mutant photolyase is photoreducable and capable of binding CPD in DNA, the activity assays indicate the mutant protein is catalytically inert. We conclude that the Asn378 residue of E. coli photolyase is crucial both for stabilizing the neutral flavin radical cofactor and for catalysis.