The initiator caspase Dronc plays a non-apoptotic role in promoting DNA damage signalling in D. melanogaster.

The phosphorylation of the variant histone H2Ax (denoted γH2Ax; γH2Av in flies) constitutes an important signalling event in DNA damage sensing, ensuring effective repair by recruiting DNA repair machinery. In contrast, the γH2Av response has also been reported in dying cells, where it requires activation of caspase-activated DNases (CADs). Moreover, caspases are known to be required downstream of DNA damage for cell death execution. We show here, for the first time, that the Drosophila initiator caspase Dronc acts as an upstream regulator of the DNA damage response (DDR) independently of executioner caspases by facilitating γH2Av signalling, possibly through a function that is not related to apoptosis. Such a γH2Av response is mediated by ATM rather than ATR, suggesting that Dronc function is required upstream of ATM. In contrast, the role of γH2Av in cell death requires effector caspases and is associated with fragmented nuclei. Our study uncovers a novel function of Dronc in response to DNA damage aimed at promoting DDR via γH2Av signalling in intact nuclei. We propose that Dronc plays a dual role that can either initiate DDR or apoptosis depending upon its level and the required threshold of its activation in damaged cells.This article has an associated First Person interview with the first author of the paper.

Chromosome territory relocation during DNA repair requires nuclear myosin 1 recruitment to chromatin mediated by ϒ-H2AX signaling.

During DNA damage response (DDR), certain gene rich chromosome territories (CTs) relocate to newer positions within interphase nuclei and revert to their native locations following repair. Such dynamic relocation of CTs has been observed under various cellular conditions, however, the underlying mechanistic basis of the same has remained largely elusive. In this study, we aim to understand the temporal and molecular details of such crosstalk between DDR signaling and CT relocation dynamics. We demonstrate that signaling at DNA double strand breaks (DSBs) by the phosphorylated histone variant (ϒ-H2AX) is a pre-requisite for damage induced CT relocation, as cells deficient in ϒ-H2AX signaling fail to exhibit such a response. Inhibition of Rad51 or DNA Ligase IV mediated late steps of double strand break repair does not seem to abrogate CT relocation completely. Upon DNA damage, an increase in the levels of chromatin bound motor protein nuclear myosin 1 (NM1) ensues, which appears to be functionally linked to ϒ-H2AX signaling. Importantly, the motor function of NM1 is essential for its recruitment to chromatin and CT relocation following damage. Taking these observations together, we propose that early DDR sensing and signaling result in NM1 recruitment to chromosomes which in turn guides DNA damage induced CT relocation.

Site-specific fluorescence dynamics in an RNA 'thermometer' reveals the role of ribosome binding in its temperature-sensitive switch function.

RNA thermometers control the translation of several heat shock and virulence genes by their temperature-sensitive structural transitions. Changes in the structure and dynamics of MiniROSE RNA, which regulates translation in the temperature range of 20-45°C, were studied by site specifically replacing seven adenine residues with the fluorescent analog, 2-aminopurine (2-AP), one at a time. Dynamic fluorescence observables of 2-AP-labeled RNAs were compared in their free versus ribosome-bound states for the first time. Noticeably, position dependence of fluorescence observables, which was prominent at 20°C, was persistent even at 45ºC, suggesting the persistence of structural integrity up to 45ºC. Interestingly, position-dependent dispersion of fluorescence lifetime and quenching constant at 45°C was ablated in ribosome-bound state, when compared to those at 20°C, underscoring loss of structural integrity at 45°C, in ribosome-bound RNA. Significant increase in the value of mean lifetime for 2-AP corresponding to Shine-Dalgarno sequences, when the temperature was raised from 20 to 45°C, to values seen in the presence of urea at 45°C was a strong indicator of melting of the 3D structure of MiniROSE RNA at 45°C, only when it was ribosome bound. Taken all together, we propose a model where we invoke that ribosome binding of the RNA thermometer critically regulates temperature sensing functions in MiniROSE RNA.

Regulation of CCM genes in Chlamydomonas reinhardtii during conditions of light-dark cycles in synchronous cultures.

We have investigated transcript level changes of CO(2)-concentrating mechanism (CCM) genes during light-dark (12 h:12 h) cycles in synchronized Chlamydomonas reinhardtii at air-level CO(2). CCM gene transcript levels vary at various times of light-dark cycles, even at same air-level CO(2). Transcripts of inorganic carbon transporter genes (HLA3, LCI1, CCP1, CCP2 and LCIA) and mitochondrial carbonic anhydrase genes (CAH4 and CAH5) are up regulated in light, following which their levels decline in dark. Contrastingly, transcripts of chloroplast carbonic anhydrases namely CAH6, CAH3 and LCIB are up regulated in dark. CAH3 and LCIB transcript levels reached maximum by the end of dark, followed by high expression into early light period. In contrast, CAH6 transcript level stayed high in dark, followed by high level even in light. Moreover, the up regulation of transcripts in dark was undone by high CO(2), suggesting that the dark induced CCM transcripts were regulated by CO(2) even in dark when CCM is absent. Thus while the CAH3 transcript level modulations appear not to positively correlate with that of CCM, the protein regulation matched with CCM status: in spite of high transcript levels in dark, CAH3 protein reached peak level only in light and localized entirely to pyrenoid, a site functionally relevant for CCM. Moreover, in dark, CAH3 protein level not only reduced but also the protein localized as a diffused pattern in chloroplast. We propose that transcription of most CCM genes, followed by protein level changes including their intracellular localization of a subset is subject to light-dark cycles.

Low energy electron induced damage to plasmid DNA pQE30.

Low energy electrons (LEEs) are produced in copious amounts by the primary radiation used in radiation therapy. The damage caused to the DNA by these secondary electrons in the energy range 5-22 eV has been studied to understand their possible role in radiation induced damage. Electrons are irradiated on dried films of plasmid DNA (pQE30) and analysed using agarose gel electrophoresis. Single strand breaks (SSBs) induced by LEE to supercoiled plasmid DNA show resonance structures at 7, 12, and 15 eV for low doses and 6, 10, and ∼18 eV at saturation doses. The present measurements have an overall agreement with the literature that LEEs resonantly induce SSBs in DNA. Resonant peaks in the SSBs induced by LEEs at 7, 12, and 15 eV with the lowest employed dose in the current study are somewhat different from those reported earlier by two groups. The observed differences are perhaps related to the irradiation dose, conditions and the nature of DNA employed, which is further elaborated.

Mammalian nuclear speckles exhibit stable association with chromatin: a biochemical study.

Nuclear Speckles (NS) are phase-separated condensates of protein and RNA whose components dynamically coordinate RNA transcription, splicing, transport and DNA repair. NS, probed largely by imaging studies, remained historically well known as Interchromatin Granule Clusters, and biochemical properties, especially their association with Chromatin have been largely unexplored. In this study, we tested whether NS exhibit any stable association with chromatin and show that limited DNAse-1 nicking of chromatin leads to the collapse of NS into isotropic distribution or aggregates of constituent proteins without affecting other nuclear structures. Further biochemical probing revealed that NS proteins were tightly associated with chromatin, extractable only by high-salt treatment just like histone proteins. NS were also co-released with solubilised mono-dinucleosomal chromatin fraction following the MNase digestion of chromatin. We propose a model that NS-chromatin constitutes a "putative stable association" whose coupling might be subject to the combined regulation from both chromatin and NS changes.Abbreviations: NS: Nuclear speckles; DSB: double strand breaks; PTM: posttranslational modifications; DDR: DNA damage repair; RBP-RNA binding proteins; TAD: topologically associated domains; LCR: low complexity regions; IDR: intrinsically disordered regions.

Fluorescence characterization of the structural heterogeneity of polytene chromosomes.

Studies on the physical nature of the structural heterogeneity of chromatin in their native states are few. The eukaryotic chromatin as observed by dye staining studies is of heterogeneous intensity when observed by fluorescent stains, where less and more bright regions apparently correspond to euchromatin and heterochromatin respectively. These are also associated with differential gene expression where it is believed that euchromatin is transcriptionally more active due to increased flexibility. Unfixed squashed preparations of polytene chromosomes of Drosophila were stained with a dsDNA specific dye PicoGreen and fluorescence lifetimes as well as fluorescence anisotropy decay kinetics were measured. Here we report a positive correlation between fluorescence lifetimes and fluorescence intensities, and show that less bright regions corresponding to euchromatin have shorter lifetimes, whereas more bright regions corresponding to heterochromatin have longer lifetimes. We interpret this as less bright regions being more dynamic, a conclusion also supported by fluorescence anisotropy decay kinetics. We infer that the comparatively higher flexibility associated with euchromatin can be directly measured by fluorescence lifetimes and fluorescence anisotropy decay kinetics.

ATR signalling mediates the prosurvival function of phospho-NPM against PIDDosome mediated cell death.

We uncover a novel non-canonical function of ATR kinase in the control of PIDDosome activation, and show that under normal cellular conditions involving no replication stress, ATR kinase controls the phosphorylation of cellular NPM via pChk1 as well as the two phosphatases, PPM1D and PP1ß. We show that pNPM triggers the dissociation of NPM from PIDD, preventing the cell from undergoing caspase 2 mediated cell death via PIDDosome, thereby acting as an endogenous negative regulator of PIDDosome activation. pChk1 interaction with NPM is abrogated following ATR kinase inhibition, leading to the drop in nucleoplasmic/chromatin pNPM level, inducing PIDD. Consistent with this mechanism, the phosphomimic mutants of Chk1 and NPM become refractory to ATR/pChk1 kinase inhibition by avoiding PIDDosome signalling.

Elucidation and genetic intervention of CO2 concentration mechanism in Chlamydomonas reinhardtii for increased plant primary productivity.

The rising global population is forcing the need for adapting alternative sustainable technologies for enhanced crop productivity. The CO2 Concentration Mechanisms (CCMs) evolved in algae to counter the inefficient CO2 fixing enzyme, RuBisCo and slower diffusion of CO2 in water offers good scope for the above purpose. The CCMs are single-celled CO2 supply mechanisms that depend on multiple CO2/HCO3 transporters and acclimation states and accumulate 100-fold more CO2 than low CO2 environments. Although some insights have been obtained regarding the CCMs of blue-green algae and green algae like Chlamydomonas reinhardtii, further progress needs to take place to understand the molecular and biochemical basis for intracellular transport of CO2. In this review, complete information pertaining to the core CCM is presented and discussed in light of the available literature. In addition to this, information on CO2/HCO3 sensing, photo-acclimation in low CO2, liquid-like nature of pyrenoid, untapped potential of high CO2 responses and high CO2 requiring mutants, and prospects of engineering CCM components into higher plants are presented and discussed.

MutS and UvrD proteins stimulate exonuclease action: insights into exonuclease-mediated strand repair.

MutS and UvrD proteins individually stimulate Escherichia coli exonuclease VII activity on blunt-ended short duplex DNA substrates. Stimulation by both proteins is ATP-dependent but not mismatch-specific and is not accompanied by apparent strand separation. Under similar conditions, MutS and UvrD proteins in fact confer resistance to exonuclease VII action on ssDNA targets, thereby implying that a novel state of a double-stranded DNA intermediate, which we term a "destabilized duplex", is involved in exonuclease-mediated strand degradation. We find that DNA strands in such a destabilized duplex can be displaced by the challenge of a molar excess of homologous single- and double-stranded DNA targets, in trans. Such an action of the UvrD protein is ATP-dependent. We discuss these results in relation to the (i) directional excision repair of E. coli MMR, (ii) downregulation of repeat deletions by exonucleases during replication slippage, and (iii) the fork reversal function of UvrD at stalled replication forks.

A comparative approach to elucidate chloroplast genome replication.

BACKGROUND: Electron microscopy analyses of replicating chloroplast molecules earlier predicted bidirectional Cairns replication as the prevalent mechanism, perhaps followed by rounds of a rolling circle mechanism. This standard model is being challenged by the recent proposition of homologous recombination-mediated replication in chloroplasts. RESULTS: We address this issue in our current study by analyzing nucleotide composition in genome regions between known replication origins, with an aim to reveal any adenine to guanine deamination gradients. These gradual linear gradients typically result from the accumulation of deaminations over the time spent single-stranded by one of the strands of the circular molecule during replication and can, therefore, be used to model the course of replication. Our linear regression analyses on the nucleotide compositions of the non-coding regions and the synonymous third codon position of coding regions, between pairs of replication origins, reveal the existence of significant adenine to guanine deamination gradients in portions overlapping the Small Single Copy (SSC) and the Large Single Copy (LSC) regions between inverted repeats. These gradients increase bi-directionally from the center of each region towards the respective ends, suggesting that both the strands were left single-stranded during replication. CONCLUSION: Single-stranded regions of the genome and gradients in time that these regions are left single-stranded, as revealed by our nucleotide composition analyses, appear to converge with the original bi-directional dual displacement loop model and restore evidence for its existence as the primary mechanism. Other proposed faster modes such as homologous recombination and rolling circle initiation could exist in addition to this primary mechanism to facilitate homoplasmy among the intra-cellular chloroplast population.

Flagella-generated forces reveal gear-type motor in single cells of the green alga, Chlamydomonas reinhardtii.

Optically trapped single cells of the biflagellated, green alga, Chlamydomonas reinhardtii, rotate. The rotational dynamics of trapped wild-type and mutant cells show that functional flagella play a decisive role: the entire flagellar apparatus (central microtubules, radial spokes, and dynein arms) is involved. Any aberration in this apparatus leads to non-functionality, indicating a gear-type mechanism. The translational and rotational motions of the wild-type and mutant cells do not differ significantly. Optical forces alone do not play a vital role in the rotational dynamics of this cellular motor, making them useful as probes of the internal dynamics without external influence.

Human Rad51 mediated DNA unwinding is facilitated by conditions that favour Rad51-dsDNA aggregation.

BACKGROUND: Human Rad51 (RAD51), analogous to its bacterial homolog, RecA, binds and unwinds double stranded DNA (dsDNA) in the presence of certain nucleotide cofactors. ATP hydrolysis is not required for this process, because even ATP non hydrolysable analogs like AMP-PNP and ATPgammaS, support DNA unwinding. Even ADP, the product of ATP hydrolysis, feebly supports DNA unwinding. RESULTS: We find that human Rad52 (RAD52) stimulates RAD51 mediated DNA unwinding in the presence of all Adenine nucleotide cofactors, (except in AMP and no nucleotide conditions that intrinsically fail to support unwinding reaction) while enhancing aggregation of RAD51-dsDNA complexes in parallel. Interestingly, salt at low concentration can substitute the role of RAD52, in facilitating aggregation of RAD51-dsDNA complexes, that concomitantly also leads to better unwinding. CONCLUSION: RAD52 itself being a highly aggregated protein perhaps acts as scaffold to bring together RAD51 and DNA molecules into large co-aggregates of RAD52-RAD51-DNA complexes to promote RAD51 mediated DNA unwinding reaction, when appropriate nucleotide cofactors are available, presumably through macromolecular crowding effects. Our work highlights the functional link between aggregation of protein-DNA complexes and DNA unwinding in RAD51 system.

Relationship between mRNA secondary structure and sequence variability in Chloroplast genes: possible life history implications.

BACKGROUND: Synonymous sites are freer to vary because of redundancy in genetic code. Messenger RNA secondary structure restricts this freedom, as revealed by previous findings in mitochondrial genes that mutations at third codon position nucleotides in helices are more selected against than those in loops. This motivated us to explore the constraints imposed by mRNA secondary structure on evolutionary variability at all codon positions in general, in chloroplast systems. RESULTS: We found that the evolutionary variability and intrinsic secondary structure stability of these sequences share an inverse relationship. Simulations of most likely single nucleotide evolution in Psilotum nudum and Nephroselmis olivacea mRNAs, indicate that helix-forming propensities of mutated mRNAs are greater than those of the natural mRNAs for short sequences and vice-versa for long sequences. Moreover, helix-forming propensity estimated by the percentage of total mRNA in helices increases gradually with mRNA length, saturating beyond 1000 nucleotides. Protection levels of functionally important sites vary across plants and proteins: r-strategists minimize mutation costs in large genes; K-strategists do the opposite. CONCLUSION: Mrna length presumably predisposes shorter mRNAs to evolve under different constraints than longer mRNAs. The positive correlation between secondary structure protection and functional importance of sites suggests that some sites might be conserved due to packing-protection constraints at the nucleic acid level in addition to protein level constraints. Consequently, nucleic acid secondary structure a priori biases mutations. The converse (exposure of conserved sites) apparently occurs in a smaller number of cases, indicating a different evolutionary adaptive strategy in these plants. The differences between the protection levels of functionally important sites for r- and K-strategists reflect their respective molecular adaptive strategies. These converge with increasing domestication levels of K-strategists, perhaps because domestication increases reproductive output.

Crystal structures of Drosophila mutant translin and characterization of translin variants reveal the structural plasticity of translin proteins.

Translin protein is highly conserved in eukaryotes. Human translin binds both ssDNA and RNA. Its nucleic acid binding site results from a combination of basic regions in the octameric structure. We report here the first biochemical characterization of wild-type Drosophila melanogaster (drosophila) translin and a chimeric translin, and present 3.5 A resolution crystal structures of drosophila P168S mutant translin from two crystal forms. The wild-type drosophila translin most likely exists as an octamer/decamer, and binds to the ssDNA Bcl-CL1 sequence. In contrast, ssDNA binding-incompetent drosophila P168S mutant translin exists as a tetramer. The structures of the mutant translin are identical in both crystal forms, and their C-terminal residues are disordered. The chimeric protein, possessing two nucleic acid binding motifs of drosophila translin, the C-terminal residues of human translin, and serine at position 168, attains the octameric state and binds to ssDNA. The present studies suggest that the oligomeric status of translin critically influences the DNA binding properties of translin proteins.

ATR kinase regulates its attenuation via PPM1D phosphatase recruitment to chromatin during recovery from DNA replication stress signalling.

In eukaryotes, in response to replication stress, DNA damage response kinase, ATR is activated, whose signalling abrogation leads to cell lethality due to aberrant fork remodelling and excessive origin firing. Here we report that inhibition of ATR kinase activity specifically during replication stress recovery results in persistent ATR signalling, evidenced by the presence of ATR-dependent phosphorylation marks (gamma H2AX, pChk1 and pRad17) and delayed cell cycle re-entry. Further, such disruption of ATR signalling attenuation leads to double-strand breaks, fork collapse and thereby 'replication catastrophe'. PPM1D phosphatase, a nucleolar localized protein, relocates to chromatin during replication stress and reverts back to nucleolus following stress recovery, under the control of ATR kinase action. Inhibition of ATR kinase activity, specifically during post replication stress, triggers dislodging of the chromatin-bound PPM1D from nucleus to cytoplasm followed by its degradation, thereby leading to persistence of activated ATR marks in the nuclei. Chemical inhibition of PPM1D activity or SiRNA mediated depletion of the protein during post replication stress recovery 'phenocopies' ATR kinase inhibition by failing to attenuate ATR signalling. Collectively, our observations suggest a novel role of ATR kinase in mediating its own signal attenuation via PPM1D recruitment to chromatin as an essential mechanism for restarting the stalled forks, cell-cycle re-entry and cellular recovery from replication stress.

Structural characterization of a novel KH-domain containing plant chloroplast endonuclease.

Chlamydomonas reinhardtii is a single celled alga that undergoes apoptosis in response to UV-C irradiation. UVI31+, a novel UV-inducible DNA endonuclease in C. reinhardtii, which normally localizes near cell wall and pyrenoid regions, gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV-stress. Solution NMR structure of the first putative UV inducible endonuclease UVI31+ revealed an α1-ß1-ß2-α2-α3-ß3 fold similar to BolA and type II KH-domain ubiquitous protein families. Three α-helices of UVI31+ constitute one side of the protein surface, which are packed to the other side, made of three-stranded ß-sheet, with intervening hydrophobic residues. A twenty-three residues long polypeptide stretch (D54-H76) connecting ß1 and ß2 strands is found to be highly flexible. Interestingly, UVI31+ recognizes the DNA primarily through its ß-sheet. We propose that the catalytic triad residues involving Ser114, His95 and Thr116 facilitate DNA endonuclease activity of UVI31+. Further, decreased endonuclease activity of the S114A mutant is consistent with the direct participation of Ser114 in the catalysis. This study provides the first structural description of a plant chloroplast endonuclease that is regulated by UV-stress response.

Expression pattern of Drosophila translin and behavioral analyses of the mutant.

Translin is an evolutionarily conserved approximately 27-kDa protein that binds to specific DNA and RNA sequences and has diverse cellular functions. Here, we report the cloning and characterization of the translin orthologue from the fruit fly Drosophila melanogaster. Under protein-denaturing conditions, purified Drosophila translin exists as a mixture of dimers and monomers just like human translin. In contrast to human translin, the Drosophila translin dimers do not appear to be stabilized by disulfide interactions. Drosophila translin shows a ubiquitous cytoplasmic localization in early embryonal syncytial stage, with an enhanced staining in ventral neuroblasts at later stages (8-9), which are probably at metaphase. An elevated expression was seen in several other cell types, such as cells around the tracheal pits in the embryo and oenocytes in the third instar larva. RNA in situ hybridization showed an increased expression in the ventral midline cells of the larval brain, suggesting a neuronal expression, which was corroborated by protein immunostaining. In adult flies, Drosophila translin is localized in the brain neuronal cell bodies and in early spermatocytes. Interestingly, Drosophila translin mutants exhibit an impaired motor response which is sex specific. Taken together, the multiple cellular localizations, the high neuronal expression and the attendant locomotor defect of the Drosophila translin mutant suggest that Drosophila translin may have roles in neuronal development and behavior analogous to that of mouse translin.

A homologue of the defender against the apoptotic death gene (dad1 )in UV-exposed Chlamydomonas cells is downregulated with the onset of programmed cell death.

We report here the isolation of a homologue of the potential anti-apoptotic gene, defender against apoptotic death (dad1 )from Chlamydomonas reinhardtii cells.Using polymerase chain reaction (PCR),we investigated its expression in the execution process of programmed cell death (PCD)in UV-C exposed dying C.reinhardtii cells.Reverse- transcriptase (RT)-PCR showed that C.reinhardtii dad1 amplification was drastically reduced in UV-C exposed dying C.reinhardtii cells.We connect the downregulation of dad1 with the upregulation of apoptosis protease activating factor-1 (APAF-1)and the physiological changes that occur in C.reinhardtii cells upon exposure to 12 J/m 2 UV-C in order to show a reciprocal relationship between proapoptotic and inhibitor of apoptosis factors.The temporal changes indicate a correlation between the onset of cell death and dad1 downregulation.The sequence of the PCR product of the cDNA encoding the dad1 homologue was aligned with the annotated dad1 (C_20215)from the Chlamydomonas database (http://genome.jgi-psf.org:8080/annotator/servlet/jgi.annotation.Annotation?pDb=chlre2); Annotation?pDb=chlre2 );this sequence was found to show 100% identity,both at the nucleotide and amino acid level. The 327 bp transcript showed an open reading frame of 87 amino acid residues.The deduced amino acid sequence of the putative C.reinhardtii DAD1 homologue showed 54% identity with Oryza sativa, 56 identity with Drosophila melanogaster, 66% identity with Xenopus laevis, and 64% identity with Homo sapiens,Sus scrofa,Gallus gallus,Rattus norvegicus and Mus musculus.

Chromosome territory relocation paradigm during DNA damage response: Some insights from molecular biology to physics.

Among the many facets of DNA damage response (DDR), relocation of chromosome territories (CTs) is most intriguing. We have previously reported that cisplatin induced DDR in human dermal fibroblasts led to relocation of CTs 12, 15 from the nuclear periphery to its interior while CTs 19, 17 repositioned from the interior to its periphery. Studies of CT relocation remain nascent as we begin unraveling the role of key players in DDR to demonstrate its mechanistic basis. Consolidating our recent reports, we argue that γH2AX-signaling leads to enhanced recruitment of nuclear myosin 1 (NM1) to chromatin, which via its motor function, results in CT repositioning. Next, we invoke a novel systems-level theory that subsumed CTs as pairs, not solo entities, to present the physical basis for plasticity in interphase CT arrangement. Subsequently, we posited that our systems-level theory describes a unified physical basis for non-random positioning of CTs in interphase nuclei across disparate eukaryotes.