American Society for Photobiology

ASP Conference 2016: 21-26 May 2016
Tampa Marriott Waterside Hotel & Marina


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15 - Frontiers in DNA Bipyrimidine Damage and Repair

Meeting Room 5   08:00 - 12:10

Chair(s): Jean Cadet
 
15-1   08:10  Combined actions of UVB and UVA on DNA damage and repair DOUKI*, Univ. Grenoble Alpes / CEA-Grenoble

Abstract: The major role played by solar UV radiation in the onset of skin cancer is mostly explained by its DNA damaging properties. An important aspect of this genotoxicity is the respective role of UVB and UVA since the proportion between the two wavelength ranges depends on the irradiation conditions and the photoprotection strategies applied. Induction of pyrimidine dimers (cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts) and oxidative lesions (oxidized bases, single strand breaks) are the most frequent DNA lesions in UV exposed cells and skin. Importantly, the nature of the photoproducts is less strictly different between UVB and UVA than previously thought as illustrated by the formation of cyclobutane dimers as the major DNA lesion in the UVA range. Data accumulated in the recent years allow us to draw a picture of the frequency of DNA damage not only after exposure to pure UVB or UVA, but also to the full complexity of sunlight. In addition, the combination of UVB and UVA has to be considered for some kind of damage (e.g. Dewar valence isomers). Modulation of the genotoxicity of UVB by UVA can also result from the alteration of the DNA repair capacities. This has been observed in both skin explants and culture keratinocytes. The latter phenomenon can be triggered by protein oxidation but may also result from difference in activated signaling pathways by either UVB or UVA. The bulk of these data has to be considered to design efficient photoprotection.

15-2   08:40  DNA repair genomics: Mapping DNA damage and DNA repair at single-nucleotide resolution across the human genome S Adar*, University of North Carolina ; J Hu, University of North Carolina; JD Lieb; A Sancar, University of North Carolina

Abstract: Ultraviolet radiation induces pyrimidine photodimers in DNA that present a barrier to transcription and replication, and compromise the ability of a cell to function. Nucleotide excision repair is the sole mechanism for removing these damages from the human genome. During human excision repair, dual incision of the damaged strand results in removal of a ~27 nucleotide-long single stranded oligomer. We have recently developed two genomic methods for mapping DNA damages and DNA repair at single nucleotide resolution across the human genome. Damages-seq relies on the replication-blocking properties of the damages to precisely map their location. In eXcision Repair-seq (XR-seq) we capture the excised oligonucleotide released during repair in vivo, and subject it to high-throughput sequencing. We used Damage-seq and XR-seq to produce genome-wide maps of two UV-induced DNA damages and follow the kinetics of their repair: cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts ((6-4)PPs). Our results show preferential repair of actively transcribed and open chromatin regions. Conversely, repair at heterochromatic and repressed regions is relatively low and continues even two days following UV irradiation. Comparing repair kinetics with existing somatic mutation data from melanoma cancer cells shows late-repaired regions are associated with a higher level of cancer-linked somatic mutations. The new genomic assays we have developed will be a powerful tool in identifying key components of genome stability, and understanding the genetic and epigenetic changes resulting from genotoxic stress.

15-3   09:10  Development of an in vivo assay to detect DNA excision repair events in mammalian cells JH Choi*, Korea Research Institute of Standards and Science, Daejeon, Korea ; MG Kemp, University of North Carolina School of Medicine; A Sancar, University of North Carolina School of Medicine

Abstract: Nucleotide excision repair is the sole DNA repair system for removing UV-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and the (6-4) photoproducts, as well as bulky base adducts induced by numerous chemical carcinogens and chemotherapeutic agents from the human genome. Studies in vitro and in vivo have shown that the UV damage is removed from the genome in the form of an oligonucleotide approximately 24- to 32-nt in length. Because there are limitations to many of the currently available methods for investigating UV photoproduct repair in vivo, we developed a convenient non-radioisotopic method to directly detect DNA excision repair events in human cells. The methodology was shown to be highly sensitive to directly detect the generation of excision products even within minutes following UV irradiation. Moreover, our techniques allowed us to examine repair events in vivo following exposure of cells to different types of DNA damaging agents causing bulky base adducts, DNA crosslinks or DNA-protein crosslinks. We suggest that the new techniques presented here will be a useful and powerful approach for studying the mechanism of human nucleotide excision repair in vivo.

15-4   09:40  Single molecule analysis of DNA repair enzymes reveals a novel "recognition at a distance" mechanism. B Van Houten*, UPCI

Abstract: Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism that processes a variety of helix-distorting lesions in DNA, such as UV-induced photoproducts. To shed light on the dynamic protein-DNA interactions during the damage recognition stage of eukaryotic NER, we employed single-molecule fluorescence microscopy, as well as atomic force microscopy (AFM). We found that quantum dot-tagged Saccharomyces cerevisiae Rad4-Rad23, once bound to UV-irradiated ï-DNA, either forms non-motile complexes or conducts one-dimensional search in two distinct diffusive modes: random linear diffusion and constrained motion. Deletion of ï¢-hairpin 3 in Rad4 resulted in increased constrained and random motion on DNA. A six amino acid deletion of the tip of ï¢-hairpin 3 increased constrained motion and was fully able to complement UV resistance of a rad4 mutant. Taken together these data indicate that Rad4 uses a novel "dynamic recognition at a distance" to identify UV photoproducts. PARP1 has recently been shown to play a role in NER and have used single molecule approaches to probe its interaction on DNA. AFM experiments indicate that PARP1 binds at nicks, AP sites, and DNA ends primarily as a monomer. Fluorescence microscopy revealed that: 1) APE1 can co-localize with PARP1; 2) APE1 diffuses more rapidly than PARP1 on DNA; 3) surprisingly, addition of NAD does not increase PARP1 dissociation from DNA; 4) PARylated PARP1 shows higher motility and less dissociation than non-PARylated PARP1; and 5) interestingly, PARP inhibitor olaparib increases PARP1 motility on AP-DNA damage arrays. Supported by NIH R01ES019566 to B.V.H.

15-5   10:30  Multiple Roles for the ATR Kinase in the Cellular Response to UV-induced DNA Damage MK Kemp*, Wright State University ; LA Lindsey-Boltz, University of North Carolina; A Sancar, University of North Carolina

Abstract: The ATR (ataxia telangiectasia and rad3-related) protein kinase regulates numerous cellular responses to ultraviolet (UV) light-induced DNA damage, including cell cycle progression, DNA replication, and apoptosis. How ATR is activated following UV irradiation to mediate specific cellular outcomes is unclear and is an important issue given that ATR has become an attractive target for cancer prevention and treatment strategies. Four main mechanisms have been proposed for ATR activation by UV-damaged DNA, including: (1) direct recognition of the damage by the kinase or associated regulatory factors; (2) the generation of single-stranded DNA gaps by nucleotide excision repair-dependent removal of UV photoproducts; (3) replicative polymerase stalling and uncoupling from DNA helicase activity during chromosomal DNA replication; and (4) interference with RNA polymerase progression during gene transcription. Our laboratory has therefore utilized a combination of biochemical and cell biological approaches to better define the activation modes and downstream functions of ATR in response to UV-induced DNA damage. For example, we have shown that ATR kinase activity is directly stimulated by the binding of ATR and its co-activator protein TopBP1 to bulky DNA adduct-containing DNA in the absence of other DNA metabolic processes. Furthermore, we recently defined a minimal set of proteins that couple the removal of DNA adducts by nucleotide excision repair and the generation of gaps in DNA to the activation of ATR. We have also characterized a variety of protein-protein and protein-DNA interactions that influence the kinetics and amplitude of ATR signaling in response to replication stress induced by DNA polymerase stalling at UV photoproducts. More recently, we have uncovered a novel pro-apoptotic function for ATR in response to UV-induced transcription stress in non-replicating cells. Our findings therefore provide critical insights into the mechanisms of ATR activation and the cellular functions of ATR in human cells exposed to UV radiation.

15-6   11:00  Cellular mechanisms of repair and response to oxidized nucleotides R Sobol*, University of South Alabama Mitchell Cancer Institute

Abstract: Abstract not available.

15-7   11:30  Effect of nucleosome formation and bending on cyclobutane dimer formation in T11-tracts K Wang, Washington University ; JS Taylor*, Washington University

Abstract: It is well established that DNA photoproducts produced by sunlight are responsible for the majority of the mutations associated with skin cancers. What is not so well established is the physical or mechanistic origin of the variation in mutation type and frequency within a gene, which must result from a complex interplay between the frequency of photoproduct formation, chemical transformation, repair, and translesion synthesis. To begin to dissect the various contributions of these factors, we have been examining the role of chromatin structure on DNA photoproduct formation and chemical transformation. It was demonstrated a number of decades ago that nucleosomes can greatly modulate cyclobutane pyrimidine dimer (CPD) formation with a 10 bp periodicity that was attributed to the effect of bending on the conformation and dynamics of DNA. Dipyrimidine sites for which the phosphodiester backbone faced out showed the greatest photoproduct yield, whereas sites facing in showed the least. The original studies of CPD modulation by nucleosomes were carried out with heterogenous nucleosomal DNA from degradation of chromatin. A number of subsequent studies with defined nucleosomal sequences did not, however, appear to show the same effect. To reinvestigate the modulation of CPD formation by nucleosomes we have studied CPD formation in T11-tracts at 7 different translational positions in a rotationally phased defined DNA sequence using a circular permutation synthesis strategy. We find that CPD formation in the T11-tracts is strongly modulated by a nucleosome, with maxima that are shifted to the 5'-side compared to heterogeneous seqences and in accord with what was previously observed for a T9-tract. The position of the maxima, however, also depended on the translational position. CPD formation was also shown to be strongly modulated in rotationally phased nucleosome-free circular DNA in the same way as found in the nucleosome, indicating that bending is the major factor in controlling CPD formation frequency.

15-8   11:50  Improvement Of UV-induced DNA Damage Repair By Chronic Low Dose Of UV MC Drigeard Desgarnier*, University Laval and Centre Hospitalier Universitaire de Quebec Research Center, Canada ; PJ Rochette, University Laval and Centre Hospitalier Universitaire de Quebec Research Center, Canada

Abstract: Ultraviolet B (UVB) is a carcinogen responsible for the induction of non-melanocytic skin cancer. UVB wavelengths exhibit their mutagenic potential by inducing two main types of DNA damage: cyclobutane pyrimidine dimer (CPD) and 6-4 photoproducts (6-4 PP). In humans, those pre-mutagenic bulky adducts are repaired by the nucleotide excision repair (NER) pathway. Since decade, the protective role of NER against skin cancer is well established; nonetheless, most of studies are based with acute dose. Few evidences in the literature are suggesting that chronic irradiation with a low sub-lethal doses of mutagen could enhance the NER capacity. However, it has never been tested whether conditioning cells with chronic low UV irradiations (CLUV) would impact NER efficiency, and the mechanisms involved in the DNA repair response after this treatment are not completely understood. Thus, we have irradiated normal human dermal fibroblasts (NHDF) with a CLUV regime, which consists of 75 J/m^2 UVB every 12 hours for 7.5 days (total 1125 J/m^2). Twelve hours following the last irradiation, cells were irradiated or not with an acute UVB dose (400 J/m^2). Our results show that CPD induced by a single acute UVB dose are 50% repaired 24h post-irradiation whereas CLUV pre-stimulated cells repaired significantly faster (70% of CPD are repaired 24h post-irradiation). The repair of 6-4PP was not improved by the CLUV pre-stimulation treatment. Our data indicate that CLUV pre-stimulation enhance the NER of CPD but not 6-4PP. Since DDB2, a DNA damage recognition protein is known to influence CPD repair efficiency but not 6-4PP, we investigate its level after the CLUV pre-stimulation. We found a 2-fold increase of DDB2 protein and mRNA levels in CLUV pre-treated cells. As DDB2 is known to be down-regulated in skin cancer, our results indicate that the CLUV treatment might have a protective effect against this neoplasia



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