Effect of Humid Air Exposed to IR Radiation on Enzyme Activity.

Water vapor absorbs well in the infra-red region of the electromagnetic spectrum. Absorption of radiant energy by water or water droplets leads to formation of exclusion zone water that possesses peculiar physico-chemical properties. In the course of this study, normally functioning and damaged alkaline phosphatase, horseradish peroxidase and catalase were treated with humid air irradiated with infrared light with a wavelength in the range of 1270 nm and referred to as coherent humidity (CoHu). One-minute long treatment with CoHu helped to partially protect enzymes from heat inactivation, mixed function oxidation, and loss of activity due to partial unfolding. Authors suggest that a possible mechanism underlying the observed effects involves altering the physicochemical properties of aqueous media while treatment of the objects with CoHu where CoHu acts as an intermediary.

The Chlamydomonas deg1c Mutant Accumulates Proteins Involved in High Light Acclimation.

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent Ser endopeptidases that perform key aspects of protein quality control in all domains of life. Here, we characterized Chlamydomonas reinhardtii DEG1C, which together with DEG1A and DEG1B is orthologous to Arabidopsis (Arabidopsis thaliana) Deg1 in the thylakoid lumen. We show that DEG1C is localized to the stroma and the periphery of thylakoid membranes. Purified DEG1C exhibited high proteolytic activity against unfolded model substrates and its activity increased with temperature and pH. DEG1C forms monomers, trimers, and hexamers that are in dynamic equilibrium. DEG1C protein levels increased upon nitrogen, sulfur, and phosphorus starvation; under heat, oxidative, and high light stress; and when Sec-mediated protein translocation was impaired. DEG1C depletion was not associated with any obvious aberrant phenotypes under nonstress conditions, high light exposure, or heat stress. However, quantitative shotgun proteomics revealed differences in the abundance of 307 proteins between a deg1c knock-out mutant and the wild type under nonstress conditions. Among the 115 upregulated proteins are PSII biogenesis factors, FtsH proteases, and proteins normally involved in high light responses, including the carbon dioxide concentrating mechanism, photorespiration, antioxidant defense, and photoprotection. We propose that the lack of DEG1C activity leads to a physiological state of the cells resembling that induced by high light intensities and therefore triggers high light protection responses.

A novel rhodopsin phosphodiesterase from Salpingoeca rosetta shows light-enhanced substrate affinity.

It is since many years textbook knowledge that the concentration of the second messenger cGMP is regulated in animal rod and cone cells by type II rhodopsins via a G-protein signaling cascade. Microbial rhodopsins with enzymatic activity for regulation of cGMP concentration were only recently discovered: in 2014 light-activated guanylyl-cyclase opsins in fungi and in 2017 a novel rhodopsin phosphodiesterase (RhoPDE) in the protist Salpingoeca rosetta (SrRhoPDE). The light regulation of SrRhoPDE, however, seemed very weak or absent. Here, we present strong evidence for light regulation by studying SrRhoPDE, expressed in Xenopus laevis oocytes, at different substrate concentrations. Hydrolysis of cGMP shows an ∼100-fold higher turnover than that of cAMP. Light causes a strong decrease in the Km value for cGMP from 80 to 13 µM but increases the maximum turnover only by ∼30%. The PDE activity for cAMP is similarly enhanced by light at low substrate concentrations. Illumination does not affect the cGMP degradation of Lys296 mutants that are not able to form a covalent bond of Schiff base type to the chromophore retinal. We demonstrate that SrRhoPDE shows cytosolic N- and C-termini, most likely via an eight-transmembrane helix structure. SrRhoPDE is a new optogenetic tool for light-regulated cGMP manipulation which might be further improved by genetic engineering.

Plant dual-specificity tyrosine phosphorylation-regulated kinase optimizes light-regulated growth and development in Arabidopsis.

Light controls vegetative and reproductive development of plants. For a plant, sensing the light input properly ensures coordination with the ever-changing environment. Previously, we found that LIGHT-REGULATED WD1 (LWD1) and LWD2 regulate the circadian clock and photoperiodic flowering. Here, we identified Arabidopsis YET ANOTHER KINASE1 (AtYAK1), an evolutionarily conserved protein and a member of dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs), as an interacting protein of LWDs. Our study revealed that AtYAK1 is an important regulator for various light responses, including the circadian clock, photomorphogenesis and reproductive development. AtYAK1 could antagonize the function of LWDs in regulating the circadian clock and photoperiodic flowering. By examining phenotypes of atyak1, we found that AtYAK1 regulated light-induced period-length shortening and photomorphogenic development. Moreover, AtYAK1 mediated plant fertility especially under inferior light conditions including low light and short-day length. This study discloses a new regulator connecting environmental light to plant growth.

Bisphosphonate inhibitors reveal a large elasticity of plastidic isoprenoid synthesis pathway in isoprene-emitting hybrid aspen.

Recently, a feedback inhibition of the chloroplastic 1-deoxy-D-xylulose 5-phosphate (DXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux.

Spatiotemporal control of cell signalling using a light-switchable protein interaction.

Genetically encodable optical reporters, such as green fluorescent protein, have revolutionized the observation and measurement of cellular states. However, the inverse challenge of using light to control precisely cellular behaviour has only recently begun to be addressed; semi-synthetic chromophore-tethered receptors and naturally occurring channel rhodopsins have been used to perturb directly neuronal networks. The difficulty of engineering light-sensitive proteins remains a significant impediment to the optical control of most cell-biological processes. Here we demonstrate the use of a new genetically encoded light-control system based on an optimized, reversible protein-protein interaction from the phytochrome signalling network of Arabidopsis thaliana. Because protein-protein interactions are one of the most general currencies of cellular information, this system can, in principle, be generically used to control diverse functions. Here we show that this system can be used to translocate target proteins precisely and reversibly to the membrane with micrometre spatial resolution and at the second timescale. We show that light-gated translocation of the upstream activators of Rho-family GTPases, which control the actin cytoskeleton, can be used to precisely reshape and direct the cell morphology of mammalian cells. The light-gated protein-protein interaction that has been optimized here should be useful for the design of diverse light-programmable reagents, potentially enabling a new generation of perturbative, quantitative experiments in cell biology.

Light induces jasmonate-isoleucine conjugation via OsJAR1-dependent and -independent pathways in rice.

The bioactive form of jasmonate is the conjugate of the amino acid isoleucine (Ile) with jasmonic acid (JA), which is biosynthesized in a reaction catalysed by the GH3 enzyme JASMONATE RESISTANT 1 (JAR1). We examined the biochemical properties of OsJAR1 and its involvement in photomorphogenesis of rice (Oryza sativa). OsJAR1 has a similar substrate specificities as its orthologue in Arabidopsis. However, osjar1 loss-of-function mutants did not show as severe coleoptile phenotypes as the JA-deficient mutants coleoptile photomorphogenesis 2 (cpm2) and hebiba, which develop long coleoptiles in all light qualities we examined. Analysis of hormonal contents in the young seedling stage revealed that osjar1 mutants are still able to synthesize JA-Ile conjugate in response to blue light, suggesting that a redundantly active enzyme can conjugate JA and Ile in rice seedlings. A good candidate for this enzyme is OsJAR2, which was found to be able to catalyse the conjugation of JA with Ile as well as with some additional amino acids. In contrast, if plants in the vegetative stage were mechanically wounded, the content of JA-Ile was severely reduced in osjar1, demonstrating that OsJAR1 is the most important JA-Ile conjugating enzyme in the wounding response during the vegetative stage.

Human exonuclease 5 is a novel sliding exonuclease required for genome stability.

Previously, we characterized Saccharomyces cerevisiae exonuclease 5 (EXO5), which is required for mitochondrial genome maintenance. Here, we identify the human homolog (C1orf176; EXO5) that functions in the repair of nuclear DNA damage. Human EXO5 (hEXO5) contains an iron-sulfur cluster. It is a single-stranded DNA (ssDNA)-specific bidirectional exonuclease with a strong preference for 5'-ends. After loading at an ssDNA end, hEXO5 slides extensively along the ssDNA prior to cutting, hence the designation sliding exonuclease. However, the single-stranded binding protein human replication protein A (hRPA) restricts sliding and enforces a unique, species-specific 5'-directionality onto hEXO5. This specificity is lost with a mutant form of hRPA (hRPA-t11) that fails to interact with hEXO5. hEXO5 localizes to nuclear repair foci in response to DNA damage, and its depletion in human cells leads to an increased sensitivity to DNA-damaging agents, in particular interstrand cross-linking-inducing agents. Depletion of hEXO5 also results in an increase in spontaneous and damage-induced chromosome abnormalities including the frequency of triradial chromosomes, suggesting an additional defect in the resolution of stalled DNA replication forks in hEXO5-depleted cells.

Production of reactive oxygen species in decoupled, Ca(2+)-depleted PSII and their use in assigning a function to chloride on both sides of PSII.

Extraction of Ca(2+) from the oxygen-evolving complex of photosystem II (PSII) in the absence of a chelator inhibits O2 evolution without significant inhibition of the light-dependent reduction of the exogenous electron acceptor, 2,6-dichlorophenolindophenol (DCPIP) on the reducing side of PSII. The phenomenon is known as "the decoupling effect" (Semin et al. Photosynth Res 98:235-249, 2008). Extraction of Cl(-) from Ca(2+)-depleted membranes (PSII[-Ca]) suppresses the reduction of DCPIP. In the current study we investigated the nature of the oxidized substrate and the nature of the product(s) of the substrate oxidation. After elimination of all other possible donors, water was identified as the substrate. Generation of reactive oxygen species HO, H2O2, and O 2 (·-) , as possible products of water oxidation in PSII(-Ca) membranes was examined. During the investigation of O 2 (·-) production in PSII(-Ca) samples, we found that (i) O 2 (·-) is formed on the acceptor side of PSII due to the reduction of O2; (ii) depletion of Cl(-) does not inhibit water oxidation, but (iii) Cl(-) depletion does decrease the efficiency of the reduction of exogenous electron acceptors. In the absence of Cl(-) under aerobic conditions, electron transport is diverted from reducing exogenous acceptors to reducing O2, thereby increasing the rate of O 2 (·-) generation. From these observations we conclude that the product of water oxidation is H2O2 and that Cl(-) anions are not involved in the oxidation of water to H2O2 in decoupled PSII(-Ca) membranes. These results also indicate that Cl(-) anions are not directly involved in water oxidation by the Mn cluster in the native PSII membranes, but possibly provide access for H2O molecules to the Mn4CaO5 cluster and/or facilitate the release of H(+) ions into the lumenal space.

The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo.

To protect cells from oxidative DNA damage and mutagenesis, organisms possess multiple glycosylases to recognize the damaged bases and to initiate the Base Excision Repair pathway. Three DNA glycosylases have been identified in mammals that are homologous to the Escherichia coli Fpg and Nei proteins, Neil1, Neil2, and Neil3. Neil1 and Neil2 in human and mouse have been well characterized while the properties of the Neil3 protein remain to be elucidated. In this study, we report the characterization of Mus musculus (house mouse) Neil3 (MmuNeil3) as an active DNA glycosylase both in vitro and in vivo. In duplex DNA, MmuNeil3 recognizes the oxidized purines, spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino- 5-formamidopyrimidine (FapyA), but not 8-oxo-7,8-dihydroguanine (8-oxoG). Interestingly, MmuNeil3 prefers lesions in single-stranded DNA and in bubble structures. In contrast to other members of the family that use the N-terminal proline as the nucleophile, MmuNeil3 forms a Schiff base intermediate via its N-terminal valine. We expressed the glycosylase domain of MmuNeil3 (MmuNeil3Delta324) in an Escherichia coli triple mutant lacking Fpg, Nei, and MutY glycosylase activities and showed that MmuNeil3 greatly reduced both the spontaneous mutation frequency and the level of FapyG in the DNA, suggesting that Neil3 plays a role in repairing FapyG in vivo.

The involvement of Arabidopsis glutathione peroxidase 8 in the suppression of oxidative damage in the nucleus and cytosol.

A family of eight genes with homology to mammalian glutathione peroxidase (GPX) isoenzymes, designated AtGPX1-AtGPX8, has been identified in Arabidopsis thaliana. In this study we demonstrated the functional analysis of Arabidopsis AtGPX8 with peroxidase activity toward H(2)O(2) and lipid hydroperoxides using thioredoxin as an electron donor. The transcript and protein levels of AtGPX8 in Arabidopsis were up-regulated coordinately in response to oxidative damage caused by high-light (HL) stress or treatment with paraquat (PQ). Furthermore, the knockout Arabidopsis mutants of AtGPX8 (KO-gpx8) exhibited increased sensitivity to oxidative damage caused by PQ treatment in root elongation compared with the wild-type plants. In contrast, transgenic lines overexpressing AtGPX8 (Ox-AtGPX8) were less sensitive to oxidative damage than the wild-type plants. The levels of oxidized proteins in the KO-gpx8 and Ox-AtGPX8 lines were enhanced and suppressed, respectively, compared with the wild-type plants under HL stress or PQ treatment. The fusion protein of AtGPX8 tagged with green fluorescent protein was localized in the cytosol and nucleus of onion epidermal cells. In addition, the AtGPX8 protein was detected in the cytosolic and nuclear fractions prepared from leaves of Arabidopsis plants using the AtGPX8 antibody. Oxidative DNA damage under treatment with PQ increased in the wild-type and KO-gpx8 plants, while it decreased in the OX-AtGPX8 plants. These results suggest that AtGPX8 plays an important role in the protection of cellular components including nuclear DNA against oxidative stress.

Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H2O2 affair?

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.

Gamma-radiation induced modifications in substrate specificity of glucose dehydrogenase and carbon source utilization pattern of phosphate-solubilizing Pantoea strains.

Glucose, maltose, and mannose as sole carbon sources, induced synthesis of glucose dehydrogenase (GDH) in three strains of Pantoea with specific activities from 0.14 to 0.6 U/mg proteins. Utilization of lactose indicated that the enzyme belongs to GDH type B isozyme. Of mutant clones, developed through radiation mutagenesis, P2-M2 utilized ribose with GDH specific activity of 0.57 U/mg protein, P4-M3 grown on glucose gave 1.5 U/mg protein and P4-M5 had high activities, when grown on galactose, maltose, and lactose. Clones P3-M2 and P2-M5 had versatile utilization of sugars and released higher amounts of P from tri-calcium phosphate and can be efficiently used for biofertilization.

Mammalian DET1 regulates Cul4A activity and forms stable complexes with E2 ubiquitin-conjugating enzymes.

DET1 (de-etiolated 1) is an essential negative regulator of plant light responses, and it is a component of the Arabidopsis thaliana CDD complex containing DDB1 and COP10 ubiquitin E2 variant. Human DET1 has recently been isolated as one of the DDB1- and Cul4A-associated factors, along with an array of WD40-containing substrate receptors of the Cul4A-DDB1 ubiquitin ligase. However, DET1 differs from conventional substrate receptors of cullin E3 ligases in both biochemical behavior and activity. Here we report that mammalian DET1 forms stable DDD-E2 complexes, consisting of DDB1, DDA1 (DET1, DDB1 associated 1), and a member of the UBE2E group of canonical ubiquitin-conjugating enzymes. DDD-E2 complexes interact with multiple ubiquitin E3 ligases. We show that the E2 component cannot maintain the ubiquitin thioester linkage once bound to the DDD core, rendering mammalian DDD-E2 equivalent to the Arabidopsis CDD complex. While free UBE2E-3 is active and able to enhance UbcH5/Cul4A activity, the DDD core specifically inhibits Cul4A-dependent polyubiquitin chain assembly in vitro. Overexpression of DET1 inhibits UV-induced CDT1 degradation in cultured cells. These findings demonstrate that the conserved DET1 complex modulates Cul4A functions by a novel mechanism.

Profiling of UV-induced ATM/ATR signaling pathways.

To ensure survival in the face of genomic insult, cells have evolved complex mechanisms to respond to DNA damage, termed the DNA damage checkpoint. The serine/threonine kinases ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) activate checkpoint signaling by phosphorylating substrate proteins at SQ/TQ motifs. Although some ATM/ATR substrates (Chk1, p53) have been identified, the lack of a more complete list of substrates limits current understanding of checkpoint pathways. Here, we use immunoaffinity phosphopeptide isolation coupled with mass spectrometry to identify 570 sites phosphorylated in UV-damaged cells, 498 of which are previously undescribed. Semiquantitative analysis yielded 24 known and 192 previously uncharacterized sites differentially phosphorylated upon UV damage, some of which were confirmed by SILAC, Western blotting, and immunoprecipitation/Western blotting. ATR-specific phosphorylation was investigated by using a Seckel syndrome (ATR mutant) cell line. Together, these results provide a rich resource for further deciphering ATM/ATR signaling and the pathways mediating the DNA damage response.

Deciphering MET-dependent modulation of global cellular responses to DNA damage by quantitative phosphoproteomics.

Increasing evidence suggests that interference with growth factor receptor tyrosine kinase (RTK) signaling can affect DNA damage response (DDR) networks, with a consequent impact on cellular responses to DNA-damaging agents widely used in cancer treatment. In that respect, the MET RTK is deregulated in abundance and/or activity in a variety of human tumors. Using two proteomic techniques, we explored how disrupting MET signaling modulates global cellular phosphorylation response to ionizing radiation (IR). Following an immunoaffinity-based phosphoproteomic discovery survey, we selected candidate phosphorylation sites for extensive characterization by targeted proteomics focusing on phosphorylation sites in both signaling networks. Several substrates of the DDR were confirmed to be modulated by sequential MET inhibition and IR, or MET inhibition alone. Upon combined treatment, for two substrates, NUMA1 S395 and CHEK1 S345, the gain and loss of phosphorylation, respectively, were recapitulated using invivo tumor models by immunohistochemistry, with possible utility in future translational research. Overall, we have corroborated phosphorylation sites at the intersection between MET and the DDR signaling networks, and suggest that these represent a class of proteins at the interface between oncogene-driven proliferation and genomic stability.

Role of the carbonyl group of the (6-4) photoproduct in the (6-4) photolyase reaction.

The (6-4) photoproduct, which is one of the major UV-induced DNA lesions, causes carcinogenesis with high frequency. The (6-4) photolyase is a flavoprotein that can restore this lesion to the original bases, but its repair mechanism has not been elucidated. In this study, we focused on the interaction between the enzyme and the 3' pyrimidone component of the (6-4) photoproduct and prepared a substrate analogue in which the carbonyl group, a hydrogen-bond acceptor, was replaced with an imine, a hydrogen-bond donor, to investigate the involvement of this carbonyl group in the (6-4) photolyase reaction. UV irradiation of oligodeoxyribonucleotides containing a single thymine-5-methylisocytosine site yielded products with absorption bands at wavelengths longer than 300 nm, similar to those obtained from the conversion of the TT site to the (6-4) photoproduct. Nuclease digestion, MALDI-TOF mass spectrometry, and the instability of the products indicated the formation of the 2-iminopyrimidine-type photoproduct. Analyses of the reaction and the binding of the (6-4) photolyase using these oligonucleotides revealed that this imine analogue of the (6-4) photoproduct was not repaired by the (6-4) photolyase, although the enzyme bound to the oligonucleotide with considerable affinity. These results indicate that the carbonyl group of the 3' pyrimidone ring plays an important role in the (6-4) photolyase reaction. On the basis of these results, we discuss the repair mechanism.

Replisome Dynamics and Their Functional Relevance upon DNA Damage through the PCNA Interactome.

Eukaryotic cells use copious measures to ensure accurate duplication of the genome. Various genotoxic agents pose threats to the ongoing replication fork that, if not efficiently dealt with, can result in replication fork collapse. It is unknown how replication fork is precisely controlled and regulated under different conditions. Here, we examined the complexity of replication fork composition upon DNA damage by using a PCNA-based proteomic screen to uncover known and unexplored players involved in replication and replication stress response. We used camptothecin or UV radiation, which lead to fork-blocking lesions, to establish a comprehensive proteomics map of the replisome under such replication stress conditions. We identified and examined two potential candidate proteins WIZ and SALL1 for their roles in DNA replication and replication stress response. In addition, our unbiased screen uncovered many prospective candidate proteins that help fill the knowledge gap in understanding chromosomal DNA replication and DNA repair.

ATR- and ATM-Mediated DNA Damage Response Is Dependent on Excision Repair Assembly during G1 but Not in S Phase of Cell Cycle.

Cell cycle checkpoint is mediated by ATR and ATM kinases, as a prompt early response to a variety of DNA insults, and culminates in a highly orchestrated signal transduction cascade. Previously, we defined the regulatory role of nucleotide excision repair (NER) factors, DDB2 and XPC, in checkpoint and ATR/ATM-dependent repair pathway via ATR and ATM phosphorylation and recruitment to ultraviolet radiation (UVR)-induced damage sites. Here, we have dissected the molecular mechanisms of DDB2- and XPC- mediated regulation of ATR and ATM recruitment and activation upon UVR exposures. We show that the ATR and ATM activation and accumulation to UVR-induced damage not only depends on DDB2 and XPC, but also on the NER protein XPA, suggesting that the assembly of an active NER complex is essential for ATR and ATM recruitment. ATR and ATM localization and H2AX phosphorylation at the lesion sites occur as early as ten minutes in asynchronous as well as G1 arrested cells, showing that repair and checkpoint-mediated by ATR and ATM starts early upon UV irradiation. Moreover, our results demonstrated that ATR and ATM recruitment and H2AX phosphorylation are dependent on NER proteins in G1 phase, but not in S phase. We reasoned that in G1 the UVR-induced ssDNA gaps or processed ssDNA, and the bound NER complex promote ATR and ATM recruitment. In S phase, when the UV lesions result in stalled replication forks with long single-stranded DNA, ATR and ATM recruitment to these sites is regulated by different sets of proteins. Taken together, these results provide evidence that UVR-induced ATR and ATM recruitment and activation differ in G1 and S phases due to the existence of distinct types of DNA lesions, which promote assembly of different proteins involved in the process of DNA repair and checkpoint activation.

Lymphocyte lineage-specific and developmental stage specific mechanisms suppress cyclin D3 expression in response to DNA double strand breaks.

Mammalian cells are thought to protect themselves and their host organisms from DNA double strand breaks (DSBs) through universal mechanisms that restrain cellular proliferation until DNA is repaired. The Cyclin D3 protein drives G1-to-S cell cycle progression and is required for proliferation of immature T and B cells and of mature B cells during a T cell-dependent immune response. We demonstrate that mouse thymocytes and pre-B cells, but not mature B cells, repress Cyclin D3 protein levels in response to DSBs. This response requires the ATM protein kinase that is activated by DSBs. Cyclin D3 protein loss in thymocytes coincides with decreased association of Cyclin D3 mRNA with the HuR RNA binding protein that ATM regulates. HuR inactivation reduces basal Cyclin D3 protein levels without affecting Cyclin D3 mRNA levels, indicating that thymocytes repress Cyclin D3 expression via ATM-dependent inhibition of Cyclin D3 mRNA translation. In contrast, ATM-dependent transcriptional repression of the Cyclin D3 gene represses Cyclin D3 protein levels in pre-B cells. Retrovirus-driven Cyclin D3 expression is resistant to transcriptional repression by DSBs; this prevents pre-B cells from suppressing Cyclin D3 protein levels and from inhibiting DNA synthesis to the normal extent following DSBs. Our data indicate that immature B and T cells use lymphocyte lineage- and developmental stage-specific mechanisms to inhibit Cyclin D3 protein levels and thereby help prevent cellular proliferation in response to DSBs. We discuss the relevance of these cellular context-dependent DSB response mechanisms in restraining proliferation, maintaining genomic integrity, and suppressing malignant transformation of lymphocytes.