Pushing the limits of flash photolysis to unravel the secrets of biological electron and proton transfer.

Time-resolved absorption spectroscopy is a powerful tool to unravel biological functions and has been a key technology for elucidating the working of electron transfer chains in photosynthesis or photorepair of UV-damaged DNA. Both of these areas have seen important contributions from laboratories all over the world, not the least of them stemming from the ingenious technical advances described by Klaus Brettel, first at the Technical University of Berlin (Germany), and later at the Atomic Energy Agency in Saclay (France). Now, after more than forty years of tireless scientific activity, Klaus is approaching retirement and this collection gathers together tributes in the form of scientific contributions from colleagues along the way, covering a spectrum of topics as diverse as photosynthesis, light-induced DNA repair, electron and proton transfer in light signalling, flavin based photo-enzymology, fluorescent marker photophysics, synthetic models and modelisation, delicate sample transient absorption spectroscopy. In an era where science is increasingly changing context from "fundamental" to "applied", Klaus' curiosity and tenacity worked hand in hand in a most effective manner to further both technical possibilities and basic understanding.

Dissecting Highly Mutagenic Processing of Complex Clustered DNA Damage in Yeast Saccharomyces cerevisiae.

Clusters of DNA damage, also called multiply damaged sites (MDS), are a signature of ionizing radiation exposure. They are defined as two or more lesions within one or two helix turns, which are created by the passage of a single radiation track. It has been shown that the clustering of DNA damage compromises their repair. Unresolved repair may lead to the formation of double-strand breaks (DSB) or the induction of mutation. We engineered three complex MDS, comprised of oxidatively damaged bases and a one-nucleotide (1 nt) gap (or not), in order to investigate the processing and the outcome of these MDS in yeast Saccharomyces cerevisiae. Such MDS could be caused by high linear energy transfer (LET) radiation. Using a whole-cell extract, deficient (or not) in base excision repair (BER), and a plasmid-based assay, we investigated in vitro excision/incision at the damaged bases and the mutations generated at MDS in wild-type, BER, and translesion synthesis-deficient cells. The processing of the studied MDS did not give rise to DSB (previously published). Our major finding is the extremely high mutation frequency that occurs at the MDS. The proposed processing of MDS is rather complex, and it largely depends on the nature and the distribution of the damaged bases relative to the 1 nt gap. Our results emphasize the deleterious consequences of MDS in eukaryotic cells.

UVB represses melanocyte cell migration and acts through ß-catenin.

The exposure of skin to ultraviolet (UV) radiation can have both beneficial and deleterious effects: it can lead, for instance, to increased pigmentation and vitamin D synthesis but also to inflammation and skin cancer. UVB may induce genetic and epigenetic alterations and have reversible effects associated with post-translational and gene regulation modifications. ß-catenin is a main driver in melanocyte development; although infrequently mutated in melanoma, its cellular localization and activity are frequently altered. Here, we evaluate the consequence of UVB on ß-catenin in the melanocyte lineage. We report that in vivo, UVB induces cytoplasmic/nuclear relocalization of ß-catenin in melanocytes of newborn mice and adult human skin. In mouse melanocyte and human melanoma cell lines in vitro, UVB increases ß-catenin stability, accumulation in the nucleus and cotranscriptional activity, leading to the repression of cell motility and velocity. The activation of the ß-catenin signalling pathway and its effect on migration by UVB are increased by an inhibitor of GSK3ß, and decreased by an inhibitor of ß-catenin. In conclusion, UVB represses melanocyte migration and does so by acting through the GSK3-ß-catenin axis.

Radiation-induced clustered DNA lesions: Repair and mutagenesis.

Clustered DNA lesions, also called Multiply Damaged Sites, is the hallmark of ionizing radiation. It is defined as the combination of two or more lesions, comprising strand breaks, oxidatively generated base damage, abasic sites within one or two DNA helix turns, created by the passage of a single radiation track. DSB clustered lesions associate DSB and several base damage and abasic sites in close vicinity, and are assimilated to complex DSB. Non-DSB clustered lesions comprise single strand break, base damage and abasic sites. At radiation with low Linear Energy Transfer (LET), such as X-rays or γ-rays clustered DNA lesions are 3-4 times more abundant than DSB. Their proportion and their complexity increase with increasing LET; they may represent a large part of the damage to DNA. Studies in vitro using engineered clustered DNA lesions of increasing complexity have greatly enhanced our understanding on how non-DSB clustered lesions are processed. Base excision repair is compromised, the observed hierarchy in the processing of the lesions within a cluster leads to the formation of SSB or DSB as repair intermediates and increases the lifetime of the lesions. As a consequence, the chances of mutation drastically increase. Complex DSB, either formed directly by irradiation or by the processing of non-DSB clustered lesions, are repaired by slow kinetics or left unrepaired and cause cell death or pass mitosis. In surviving cells, large deletions, translocations, and chromosomal aberrations are observed. This review details the most recent data on the processing of non-DSB clustered lesions and complex DSB and tends to demonstrate the high significance of these specific DNA damage in terms of genomic instability induction.

Singlet Oxygen-Mediated Oxidation during UVA Radiation Alters the Dynamic of Genomic DNA Replication.

UVA radiation (320-400 nm) is a major environmental agent that can exert its deleterious action on living organisms through absorption of the UVA photons by endogenous or exogenous photosensitizers. This leads to the production of reactive oxygen species (ROS), such as singlet oxygen (1O2) and hydrogen peroxide (H2O2), which in turn can modify reversibly or irreversibly biomolecules, such as lipids, proteins and nucleic acids. We have previously reported that UVA-induced ROS strongly inhibit DNA replication in a dose-dependent manner, but independently of the cell cycle checkpoints activation. Here, we report that the production of 1O2 by UVA radiation leads to a transient inhibition of replication fork velocity, a transient decrease in the dNTP pool, a quickly reversible GSH-dependent oxidation of the RRM1 subunit of ribonucleotide reductase and sustained inhibition of origin firing. The time of recovery post irradiation for each of these events can last from few minutes (reduction of oxidized RRM1) to several hours (replication fork velocity and origin firing). The quenching of 1O2 by sodium azide prevents the delay of DNA replication, the decrease in the dNTP pool and the oxidation of RRM1, while inhibition of Chk1 does not prevent the inhibition of origin firing. Although the molecular mechanism remains elusive, our data demonstrate that the dynamic of replication is altered by UVA photosensitization of vitamins via the production of singlet oxygen.

Fission yeast Rad52 phosphorylation restrains error prone recombination pathways.

Rad52 is a key protein in homologous recombination (HR), a DNA repair pathway dedicated to double strand breaks and recovery of blocked or collapsed replication forks. Rad52 allows Rad51 loading on single strand DNA, an event required for strand invasion and D-loop formation. In addition, Rad52 functions also in Rad51 independent pathways because of its ability to promote single strand annealing (SSA) that leads to loss of genetic material and to promote D-loops formation that are cleaved by Mus81 endonuclease. We have previously reported that fission yeast Rad52 is phosphorylated in a Sty1 dependent manner upon oxidative stress and in cells where the early step of HR is impaired because of lack of Rad51. Here we show that Rad52 is also constitutively phosphorylated in mus81 null cells and that Sty1 partially impinges on such phosphorylation. As upon oxidative stress, the Rad52 phosphorylation in rad51 and mus81 null cells appears to be independent of Tel1, Rad3 and Cdc2. Most importantly, we show that mutating serine 365 to glycine (S365G) in Rad52 leads to loss of the constitutive Rad52 phosphorylation observed in cells lacking Rad51 and to partial loss of Rad52 phosphorylation in cells lacking Mus81. Contrariwise, phosphorylation of Rad52-S365G protein is not affected upon oxidative stress. These results indicate that different Rad52 residues are phosphorylated in a Sty1 dependent manner in response to these distinct situations. Analysis of spontaneous HR at direct repeats shows that mutating serine 365 leads to an increase in spontaneous deletion-type recombinants issued from mitotic recombination that are Mus81 dependent. In addition, the recombination rate in the rad52-S365G mutant is further increased by hydroxyurea, a drug to which mutant cells are sensitive.

Oxidative stress in mammalian cells impinges on the cysteines redox state of human XRCC3 protein and on its cellular localization.

In vertebrates, XRCC3 is one of the five Rad51 paralogs that plays a central role in homologous recombination (HR), a key pathway for maintaining genomic stability. While investigating the potential role of human XRCC3 (hXRCC3) in the inhibition of DNA replication induced by UVA radiation, we discovered that hXRCC3 cysteine residues are oxidized following photosensitization by UVA. Our in silico prediction of the hXRCC3 structure suggests that 6 out of 8 cysteines are potentially accessible to the solvent and therefore potentially exposed to ROS attack. By non-reducing SDS-PAGE we show that many different oxidants induce hXRCC3 oxidation that is monitored in Chinese hamster ovarian (CHO) cells by increased electrophoretic mobility of the protein and in human cells by a slight decrease of its immunodetection. In both cell types, hXRCC3 oxidation was reversed in few minutes by cellular reducing systems. Depletion of intracellular glutathione prevents hXRCC3 oxidation only after UVA exposure though depending on the type of photosensitizer. In addition, we show that hXRCC3 expressed in CHO cells localizes both in the cytoplasm and in the nucleus. Mutating all hXRCC3 cysteines to serines (XR3/S protein) does not affect the subcellular localization of the protein even after exposure to camptothecin (CPT), which typically induces DNA damages that require HR to be repaired. However, cells expressing mutated XR3/S protein are sensitive to CPT, thus highlighting a defect of the mutant protein in HR. In marked contrast to CPT treatment, oxidative stress induces relocalization at the chromatin fraction of both wild-type and mutated protein, even though survival is not affected. Collectively, our results demonstrate that the DNA repair protein hXRCC3 is a target of ROS induced by environmental factors and raise the possibility that the redox environment might participate in regulating the HR pathway.

Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters.

Unresolved repair of clustered DNA lesions can lead to the formation of deleterious double strand breaks (DSB) or to mutation induction. Here, we investigated the outcome of clusters composed of base lesions for which base excision repair enzymes have different kinetics of excision/incision. We designed multiply damaged sites (MDS) composed of a rapidly excised uracil (U) and two oxidized bases, 5-hydroxyuracil (hU) and 8-oxoguanine (oG), excised more slowly. Plasmids harboring these U-oG/hU MDS-carrying duplexes were introduced into Escherichia coli cells either wild type or deficient for DNA n-glycosylases. Induction of DSB was estimated from plasmid survival and mutagenesis determined by sequencing of surviving clones. We show that a large majority of MDS is converted to DSB, whereas almost all surviving clones are mutated at hU. We demonstrate that mutagenesis at hU is correlated with excision of the U placed on the opposite strand. We propose that excision of U by Ung initiates the loss of U-oG-carrying strand, resulting in enhanced mutagenesis at the lesion present on the opposite strand. Our results highlight the importance of the kinetics of excision by base excision repair DNA n-glycosylases in the processing and fate of MDS and provide evidence for the role of strand loss/replication fork collapse during the processing of MDS on their mutational consequences.

Stress activated protein kinase pathway modulates homologous recombination in fission yeast.

Rad52 is a key player in homologous recombination (HR), a DNA repair pathway that is dedicated to double strand breaks repair and recovery of perturbed replication forks. Here we show that fission yeast Rad52 homologue is phosphorylated when S phase cells are exposed to ROS inducers such as ultraviolet A radiation or hydrogen peroxide, but not to ultraviolet C or camptothecin. Phosphorylation does not depend on kinases Chk1, Rad3, Tel1 or Cdc2, but depends on a functional stress activated protein kinase (SAPK) pathway and can be partially prevented by anti-oxidant treatment. Indeed, cells lacking Sty1, the major fission yeast MAP kinase of the SAPK pathway, do not display Rad52 phosphorylation and have UVA induced Rad52 foci that persist longer if compared to wild type cells. In addition, spontaneous intrachromosomal HR is diminished in cells lacking Sty1 and, more precisely, gene conversion is affected. Moreover, HR induced by site-specific arrest of replication forks is twice less efficient in cells that do not express Sty1. Importantly, impairing HR by deletion of the gene encoding the recombinase Rhp51 leads to Sty1 dependent Rad52 phosphorylation. Thus, SAPK pathway impinges on early step of HR through phosphorylation of Rad52 in cells challenged by oxidative stress or lacking Rhp51 and is required to promote spontaneous gene conversion and recovery from blocked replication forks.

Unravelling UVA-induced mutagenesis.

Ultraviolet A (UVA) radiation represents more than 90% of the solar UV radiation reaching Earth's surface. Exposure to solar UV radiation is a major risk in the occurrence of non-melanoma skin cancer. Whole genome sequencing data of melanoma tumors recently obtained makes it possible also to definitively associate malignant melanoma with sunlight exposure. Even though UVB has long been established as the major cause of skin cancer, the relative contribution of UVA is still unclear. In this review, we first report on the formation of DNA damage induced by UVA radiation, and on recent advances on the associated mechanism. We then discuss the controversial data on the UVA-induced mutational events obtained for various types of eukaryotic cells, including human skin cells. This may help unravel the role of UVA in the various steps of photocarcinogenesis. The connection to photocarcinogenesis is more extensively discussed by other authors in this issue.

Clustered DNA lesion repair in eukaryotes: relevance to mutagenesis and cell survival.

A clustered DNA lesion, also known as a multiply damaged site, is defined as ≥ 2 damages in the DNA within 1-2 helical turns. Only ionizing radiation and certain chemicals introduce DNA damage in the genome in this non-random way. What is now clear is that the lethality of a damaging agent is not just related to the types of DNA lesions introduced, but also to how the damage is distributed in the DNA. Clustered DNA lesions were first hypothesized to exist in the 1990s, and work has progressed where these complex lesions have been characterized and measured in irradiated as well as in non-irradiated cells. A clustered lesion can consist of single as well as double strand breaks, base damage and abasic sites, and the damages can be situated on the same strand or opposing strands. They include tandem lesions, double strand break (DSB) clusters and non-DSB clusters, and base excision repair as well as the DSB repair pathways can be required to remove these complex lesions. Due to the plethora of oxidative damage induced by ionizing radiation, and the repair proteins involved in their removal from the DNA, it has been necessary to study how repair systems handle these lesions using synthetic DNA damage. This review focuses on the repair process and mutagenic consequences of clustered lesions in yeast and mammalian cells. By examining the studies on synthetic clustered lesions, and the effects of low vs high LET radiation on mammalian cells or tissues, it is possible to extrapolate the potential biological relevance of these clustered lesions to the killing of tumor cells by radiotherapy and chemotherapy, and to the risk of cancer in non-tumor cells, and this will be discussed.

Photosensitization induced by the antibacterial fluoroquinolone Rufloxacin leads to mutagenesis in yeast.

Rufloxacin (RFX) is an antibacterial fluoroquinolone that exhibits UVA photosensitization properties. Photosensitization reactions lead to the formation of oxidative damage, mainly via singlet oxygen. Here we explore the phototoxic and photomutagenic potency of RFX using a panel of yeast (Saccharomyces cerevisiae) mutants affected in different DNA repair pathways. Yeast mutants provide a sensitive tool to identify the photodamage and the DNA repair pathways that cope with it. Cell viability test at increasing dose of UVA shows that both the DNA repair deficient and wild type cells are equally sensitive to RFX-induced photosensitization, demonstrating that phototoxic effect is not due to DNA injury. Photomutagenicity of RFX is evaluated by measuring the frequency of forward Can(R) mutations. The mutation induction is low in wild type cells. A high increase in mutation frequency is observed in strains affected in Ogg1 gene, compared to wild type and other base excision repair deficient strains. The mutation spectrum photomediated by RFX in wild type cells reveals a bias in favour of GC>TA transversions, whereas transition and frameshift mutations are less represented. Altogether data demonstrates that 8-oxo-7,8-dihydroguanine (8-oxoGua) is by far the major DNA damage produced by RFX photosensitization, leading to mutagenesis. We also explore the role played by DNA mismatch repair, translesion synthesis and post-replication repair in the prevention of mutagenic effects due to RFX exposure. In addition, we show that most of RFX photodegradation products are not mutagenic. This study defines the phototoxic and photomutagenic properties of antibacterial RFX and point out possible unwanted side effects in skin under sunlight.

Double-strand break induction and repair in V79-4 hamster cells: the role of core ionisations, as probed by ultrasoft X-rays.

PURPOSE: To compare the induction of double-strand breaks (DSB) in cells irradiated by 250 and 350 eV ultrasoft X-rays and assess the residual yield of breaks 2 hours post irradiation in order to unravel the correlation between the sharp increase in cell-killing efficiency of ultrasoft X-rays above versus below the carbon-K threshold (284 eV) and the induction of core events in DNA atoms. MATERIALS AND METHODS: V79-4 hamster cells were irradiated with synchrotron ultrasoft X-rays at isoattenuating energies of 250 eV and 350 eV. DSB were quantified using pulse field gel electrophoresis. RESULTS: A significant increase in DSB induction was observed for 350 eV ultrasoft X-rays above the carbon-K threshold, compared to 250 eV below the threshold, per unit dose to the cell. The DSB induced by the 350 eV ultrasoft X-rays were less repaired 2 h after irradiation. CONCLUSION: The increased DSB induction at 350 eV is attributed to the increase in the relative proportion of photon interactions in DNA resulting in significant dose inhomogeneity across the cell with a local increase in dose to DNA. It results from an increase in carbon-K shell interactions and the short range of the electrons produced. Core ionisations in DNA, through core-hole relaxation in conjunction with localised effects of spatially correlated low-energy photo- and Auger-electrons lead to an increase in number and the complexity of DSB.

The formation of double-strand breaks at multiply damaged sites is driven by the kinetics of excision/incision at base damage in eukaryotic cells.

It has been stipulated that repair of clustered DNA lesions may be compromised, possibly leading to the formation of double-strand breaks (DSB) and, thus, to deleterious events. Using a variety of model multiply damaged sites (MDS), we investigated parameters that govern the formation of DSB during the processing of MDS. Duplexes carrying MDS were inserted into replicative or integrative vectors, and used to transform yeast Saccharomyces cerevisiae. Formation of DSB was assessed by a relevant plasmid survival assay. Kinetics of excision/incision and DSB formation at MDS was explored using yeast cell extracts. We show that MDS composed of two uracils or abasic sites, were rapidly incised and readily converted into DSB in yeast cells. In marked contrast, none of the MDS carrying opposed oG and hU separated by 3-8 bp gave rise to DSB, despite the fact that some of them contained preexisting single-strand break (a 1-nt gap). Interestingly, the absence of DSB formation in this case correlated with slow excision/incision rates of lesions. We propose that the kinetics of the initial repair steps at MDS is a major parameter that direct towards the conversion of MDS into DSB. Data provides clues to the biological consequences of MDS in eukaryotic cells.

Induction and repairability of DNA damage caused by ultrasoft X-rays: role of core events.

PURPOSE: To investigate the severity of damage induced in plasmid DNA by ultrasoft X-rays at different energies, in order to unravel the correlation between the sharp increase in cell-killing efficiency of ultrasoft X-rays above versus below the carbon K-threshold and the induction of core events in DNA atoms. MATERIALS AND METHODS: Bluescript (pBS, tight packing) and pSP189 (pSP, loose packing) plasmids were exposed to ultrasoft X-rays at 250, 380 and 760 eV energies, respectively, above phosphorus L-, carbon K- and oxygen K-thresholds. Complex DNA lesions were assayed by the repair protein Formamidopyrimidine DNA glycosylase (Fpg) and by in vitro repair assay using whole cell-free extracts. RESULTS: Clustered damage, as revealed by Fpg-induced double strand breaks, was observed at low level, but at similar rate at the three energies. Damage induced at 380 eV may be slightly less efficiently repaired by cell extracts than those produced at 250 eV. 760 eV photons which yield longer range electrons than 250 and 380 eV photons, induced more total damages which were more efficiently repaired, and thus likely more dispersed. CONCLUSION: It is demonstrated that ultrasoft X-rays induce complex damage, which do not exhibit the same ability to be repaired, depending on the energy and on DNA packing.

Inhibition of S-phase progression triggered by UVA-induced ROS does not require a functional DNA damage checkpoint response in mammalian cells.

Ultraviolet A (UVA) radiation represents more than 90% of the UV spectrum reaching Earth's surface. Exposure to UV light, especially the UVA part, induces the formation of photoexcited states of cellular photosensitizers with subsequent generation of reactive oxygen species (ROS) leading to damages to membrane lipids, proteins and nucleic acids. Although UVA, unlike UVC and UVB, is poorly absorbed by DNA, it inhibits cell cycle progression, especially during S-phase. In the present study, we examined the role of the DNA damage checkpoint response in UVA-induced inhibition of DNA replication. We provide evidence that UVA delays S-phase in a dose dependent manner and that UVA-irradiated S-phase cells accumulate in G2/M. We show that upon UVA irradiation ATM-, ATR- and p38-dependent signalling pathways are activated, and that Chk1 phosphorylation is ATR/Hus1 dependent while Chk2 phosphorylation is ATM dependent. To assess for a role of these pathways in UVA-induced inhibition of DNA replication, we investigated (i) cell cycle progression of BrdU labelled S-phase cells by flow cytometry and (ii) incorporation of [methyl-(3)H]thymidine, as a marker of DNA replication, in ATM, ATR and p38 proficient and deficient cells. We demonstrate that none of these pathways is required to delay DNA replication in response to UVA, thus ruling out a role of the canonical S-phase checkpoint response in this process. On the contrary, scavenging of UVA-induced reactive oxygen species (ROS) by the antioxidant N-acetyl-L-cystein or depletion of vitamins during UVA exposure significantly restores DNA synthesis. We propose that inhibition of DNA replication is due to impaired replication fork progression, rather as a consequence of UVA-induced oxidative damage to protein than to DNA.

Unconventional effects of UVA radiation on cell cycle progression in S. pombe.

UVA radiation, the most abundant solar UV radiation reaching Earth's surface, induces oxidative stress through formation of reactive oxygen species (ROS) that can damage different cell components. Because of the broad spectrum of the possible targets of ROS, the cellular response to this radiation is complex. While extensive studies have allowed dissecting the effects of UVB, UVC and gamma radiations on cell cycle progression, few studies have dealt with the effect of UVA so far. Here we use Schizosaccharomyces pombe as a model organism to study biological effects of UVA radiation in living organisms. Through analysis of cell cycle progression in different mutant backgrounds we demonstrate that UVA delays cell cycle progression in G(2) cells in a dose dependent manner. However, despite Chk1 phosphorylation and in contrast to treatments with others genotoxic agents, this cell cycle delay is only partially dependent on DNA integrity checkpoint pathway. We also demonstrate that UVA irradiation of S phase cells slows down DNA replication in a checkpoint independent manner, activates Chk1 to prevent entry into abnormal mitosis and induces formation of Rad22 (homologue to human Rad52) foci. This indicates that DNA structure integrity is challenged. Furthermore, the cell cycle delay observed in checkpoint mutants exposed to UVA is not abolished when stress response pathway is inactivated or when down regulation of protein synthesis is prevented. In conclusion, fission yeast is a useful model to dissect the fundamental molecular mechanisms involved in UVA response that may contribute to skin cancer and aging.