DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein

[EN] Single-stranded DNA-binding proteins (SSBs) play a key role in genome maintenance, binding and organizing single-stranded DNA (ssDNA) intermediates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), present multiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentration. It has long been suggested that different binding modes might be used selectively for different functions. To study this possibility, we used optical tweezers to determine and compare the structure and energetics of long, individual HmtSSB¿DNA complexes assembled on preformed ssDNA and on ssDNA generated gradually during `in situ¿ DNA synthesis. We show that HmtSSB binds to preformed ss-DNA in two major modes, depending on salt and protein concentration. However, when protein binding was coupled to strand-displacement DNA synthesis, only one of the two binding modes was observed under all experimental conditions. Our results reveal a key role for the gradual generation of ssDNA in modulating the binding mode of a multimeric SSB protein and consequently, in generating the appropriate nucleoprotein structure for DNA synthetic reactions required for genome maintenance.We are grateful to Prof. M. Salas laboratory (CBMSO-CSIC) for generously providing the Phi29 DNA polymerase and to Juan P. García Villaluenga (UCM) for useful discussions. Spanish Ministry of Economy and Competitiveness [MAT2015-71806-R to J.R.A-G, FIS2010-17440, FIS2015-67765-R to F.J.C., BFU2012-31825, BFU2015-63714-R to B.I.]; Spanish Ministry of Education, Culture and Sport [FPU13/02934 to J.J., FPU13/02826 to E.B-H.]; National Institutes of Health [GM45925 to L.S.K.]; University of Tampere (to G.L.C.); Programa de Financiacion Universidad Complutense de Madrid-Santander Universidades [CT45/15-CT46/15 to F.C.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2015-63714-R].Morin, J.; Cerrón, F.; Jarillo, J.; Beltran-Heredia, E.; Ciesielski, G.; Arias-Gonzalez, JR.; Kaguni, L.... (2017). DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein. Nucleic Acids Research. 45(12):7237-7248. https://doi.org/10.1093/nar/gkx395S723772484512Shereda, R. D., Kozlov, A. G., Lohman, T. M., Cox, M. M., & Keck, J. L. (2008). 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Revisión. Control fotoperiódico del tiempo de floración

The rotation of the earth results in periodic changes in environmental factors such as daylength and temperature; the circadian clock is the endogenous mechanism responsible for day-length measurement, and allows plants to anticipate these fluctuations and modulate their developmental programs to maximize adaptation to those environmental cues. Flowering represents the transition from a vegetative to reproductive phase and is controlled by complex and highly regulated genetic pathways. In many plants, the time of flowering is strongly influenced by photoperiod, which synchronizes the floral transition with the favourable season of the year. Over the last decade, genetic approaches have aided the discovery of many signalling components involved in the photoperiod pathway and here, we highlight the significant progress made in identifying the molecular mechanisms that measure daylength and control flowering initiation in Arabidopsis, a long day (LD) plant, and in rice, a short day (SD) plant. Some components of the Arabidopsis regulatory network are conserved in other species, but the difference in the function of particular genes may contribute to the opposite photoperiodic flowering response observed between LD and SD plants. The specific regulatory mechanisms involved in controlling CONSTANS (CO) expression and stability by the circadian clock and the different photoreceptors will be described. In addition, the role of FLOWERING LOCUS T (FT), as part of the florigen, and several other light signalling and circadian-dependent components in photoperiodic flowering will be also discussed.La rotación diaria de la tierra provoca cambios periódicos en la duración del día o en la temperatura, y el reloj circadiano es un mecanismo endógeno responsable de la medida de la duración del día; este oscilador molecular permite a los organismos anticiparse a dichos cambios y adaptar su desarrollo de manera adecuada. La floración representa la transición desde una fase vegetativa del crecimiento a una reproductiva, y está controlada por diferentes rutas, muy complejas y altamente reguladas. En muchas plantas, esta transición está controlada principalmente por la duración del día o fotoperiodo, el cual sincroniza la floración con la estación más favorable del año. En la última década, diferentes estudios genéticos han facilitado la caracterización de muchos componentes de señalización involucrados en la ruta del fotoperiodo y en esta revisión se discute el progreso reciente que se ha llevado a cabo en la identificación de los mecanismos moleculares que permiten medir la duración del día, y que controlan el tiempo de floración en Arabidopsis, una especie de día largo, y en arroz, un especie de día corto. Aunque los componentes de las rutas reguladoras de Arabidopsis parecen conservarse en otras especies, la diferencia de función de genes concretos parece contribuir a la respuesta opuesta a la duración del día observada entre especies de día largo y de día corto. Se describen los mecanismos reguladores específicos que participan en la expresión y estabilidad de CONSTANS (CO) por el reloj circadiano y por diferentes fotorreceptores. Además, también discutiremos el papel del locus FLOWERING LOCUS T (FT), como parte del florígeno, y de otros componentes dependientes del mecanismo del reloj circadiano o de la señalización por luz en el control por fotoperiodo del tiempo de floración

Chromatin-dependent repression of the Arabidopsis floral integrator genes involves plant specific PHD-containing proteins

The interplay among histone modifications modulates the expression of master regulatory genes in development. Chromatin effector proteins bind histone modifications and translate the epigenetic status into gene expression patterns that control development. Here, we show that two Arabidopsis thaliana paralogs encoding plant-specific proteins with a plant homeodomain (PHD) motif, SHORT LIFE (SHL) and EARLY BOLTING IN SHORT DAYS (EBS), function in the chromatin-mediated repression of floral initiation and play independent roles in the control of genes regulating flowering. Previous results showed that repression of the floral integrator FLOWERING LOCUS T (FT) requires EBS. We establish that SHL is necessary to negatively regulate the expression of SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), another floral integrator. SHL and EBS recognize di- and trimethylated histone H3 at lysine 4 and bind regulatory regions of SOC1 and FT, respectively. These PHD proteins maintain an inactive chromatin conformation in SOC1 and FT by preventing high levels of H3 acetylation, bind HISTONE DEACETYLASE6, and play a central role in regulating flowering time. SHL and EBS are widely conserved in plants but are absent in other eukaryotes, suggesting that the regulatory module mediated by these proteins could represent a distinct mechanism for gene expression control in plants

Early in short days 1 (ESD1) encodes Actin-Related Protein 6 (AtARP6), a putative component of chromatin remodelling complexes that positively regulates FLC accumulation in Arabidopsis

We have characterized Arabidopsis esd1 mutations, which cause early flowering independently of photoperiod, moderate increase of hypocotyl length, shortened inflorescence internodes, and altered leaf and flower development. Phenotypic analyses of double mutants with mutations at different loci of the flowering inductive pathways suggest that esd1 abolishes the FLC-mediated late flowering phenotype of plants carrying active alleles of FRI and of mutants of the autonomous pathway. We found that ESD1 is required for the expression of the FLC repressor to levels that inhibit flowering. However, the effect of esd1 in a flc-3 null genetic background and the downregulation of other members of the FLC-like/MAF gene family in esd1 mutants suggest that flowering inhibition mediated by ESD1 occurs through both FLC- and FLC-like gene-dependent pathways. The ESD1 locus was identified through a map-based cloning approach. ESD1 encodes ARP6, a homolog of the actin-related protein family that shares moderate sequence homology with conventional actins. Using chromatin immunoprecipitation (ChlP) experiments, we have determined that ARP6 is required for both histone acetylation and methylation of the FLC chromatin in Arabidopsis

Low temperature regulates Arabidopsis Lhcb gene expression in a light-independent manner

Low temperature treatment of dark-grown seedlings of Arabidopsis thaliana results in a rapid increase in the amount of mRNAs encoding for the major polypeptides of the light-harvesting complex of photosystem II (Lhcb1 genes). This increase is transient and seems to be due mainly to the accumulation of Lhcb1*3 transcripts, indicating that low temperature differentially regulates the expression of the Arabidopsis Lhcb1 gene family in the dark. A 1.34 kb fragment of the Lhcb1*3 promoter is sufficient to confer low temperature regulation to a reporter gene in transgenic Arabidopsis etiolated seedlings, suggesting that the regulation is occurring at the transcriptional level. The cold-induced accumulation of Lhcb1*3 mRNA is not part of a general response to stressful conditions since no accumulation is detected in response to water stress, anaerobiosis or salt stress. The amount of Lhcb1*3 mRNA decrease in response to exogenous abscisic acid (ABA) suggesting that this phytohormone acts as a negative regulator. Moreover, the accumulation of Lhcb1*3 mRNAs in cold-treated ABA deficient etiolated seedlings is higher than that of wild-type and ABA insensitive etiolated seedlings, indicating that low temperature regulation of Lhcb1*3 is not mediated by ABA

A search for homologues of plant photoreceptor genes and their signaling partners in the sugarcane expressed sequence tag (Sucest) database

A search in the sugarcane expressed sequence tag (SUCEST) database for homologues of plant genes involved in photo-sensory mechanisms was carried out using the basic local alignment tool (BLAST). Our results shown that known elements (phytochromes, cryptochromes and phototoprin) present in Arabidopsis and other higher plants were detected with low e-values. We also searched for proteins interacting with photoreceptors in primary or downstream signaling events. One putative homologue for a protein postulated to be a primary element in phytochrome signaling pathways was identified, as were other candidates for downstream interacting factors.<br>A partir dos dados do projeto de sequenciamento de Ests da Cana de Açúcar (Sucest/FAPESP) e utilizando BLAST (tblastn) como ferramenta, foi realizada uma busca de genes homólogos aos elementos envolvidos nos processos de foto-recepção e já descritos para outras plantas, principalmente Arabidopsis. Foram obtidas altas identidades para os fitocromos A, B e C assim como para os criptocromos 1, 2 e a fototropina. Diversos elementos identificados como reguladores primários ou secundários na transdução de sinal de foto-receptores também foram identificados com baixos valores de E-value