Subtelomeric I-scel-mediated double-strand breaks are repaired by homologous recombination in trypanosoma cruzi

Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a I cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-Scel meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families7CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP306591/2015-411/51693-0; 11/51475-

The arabidopsis DNA polymerase δ has a role in the deposition of transcriptionally active epigenetic marks, development and flowering

DNA replication is a key process in living organisms. DNA polymerase α (Polα) initiates strand synthesis, which is performed by Polε and Polδ in leading and lagging strands, respectively. Whereas loss of DNA polymerase activity is incompatible with life, viable mutants of Polα and Polε were isolated, allowing the identification of their functions beyond DNA replication. In contrast, no viable mutants in the Polδ polymerase-domain were reported in multicellular organisms. Here we identify such a mutant which is also thermosensitive. Mutant plants were unable to complete development at 28°C, looked normal at 18°C, but displayed increased expression of DNA replication-stress marker genes, homologous recombination and lysine 4 histone 3 trimethylation at the SEPALLATA3 (SEP3) locus at 24°C, which correlated with ectopic expression of SEP3. Surprisingly, high expression of SEP3 in vascular tissue promoted FLOWERING LOCUS T (FT) expression, forming a positive feedback loop with SEP3 and leading to early flowering and curly leaves phenotypes. These results strongly suggest that the DNA polymerase δ is required for the proper establishment of transcriptionally active epigenetic marks and that its failure might affect development by affecting the epigenetic control of master genes.Fil: Iglesias, Francisco Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Bruera, Natalia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Dergan Dylon, Leonardo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Marino, Cristina Ester. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Lorenzi, Hernán. J. Craig Venter Institute; Estados UnidosFil: Mateos, Julieta Lisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Turck, Franziska. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Coupland, George. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Cerdan, Pablo Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Universidad de Buenos Aires. Departamento de Ciencias Exactas; Argentin

<i>gis5</i> phenotypes are temperature-dependent.

<p>(<i>A</i>) <i>gis5</i> mutants flower early. WT, <i>gis5</i> and <i>gi-2</i> single mutants and <i>gis5 gi-2</i> double mutant lines were grown under LD at 23°C. The total leaf number (cauline and rosette leaves) was recorded at the time of flowering. Bars represent the mean ± SEM of at least 12 plants for each genotype. (<i>B</i> and <i>C</i>) <i>gis5</i> mutants display a temperature-dependent curly leaf phenotype. WT and <i>gis5</i> mutants were grown under LD at the indicated temperatures either on soil (<i>B</i>) or MS agar plates (<i>C</i>) and photographed at flowering. Scale bar: 1 cm. (<i>D</i> and <i>E</i>) <i>gis5</i> mutants display a temperature-dependent early flowering phenotype under SD. WT and <i>gis5</i> mutants were grown under LD (<i>D</i>) or SD (<i>E</i>) at the indicated temperatures (abscissas) and flowering time was recorded as in (<i>A</i>). Bars represent the mean ± SEM of at least 12 plants for each genotype.</p

High levels of <i>SEP3</i> expression are required for <i>gis5</i> early flowering and leaf phenotype.

<p>(<i>A</i>) <i>SEP3</i> downregulation delays flowering in <i>gis5</i> mutants. Plants of the genotypes indicated in coloured bars were grown under either LD (left panel) or SD (right panel) at 23°C. The total leaf number (cauline and rosette leaves) was recorded at the time of flowering. Bars represent the mean ± SEM of at least 12 plants for each genotype. (<i>B</i>) <i>SEP3</i> and <i>SEP1</i> mRNA expression levels were determined in the same genotypes shown in (<i>A</i>) as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004975#pgen.1004975.g005" target="_blank">Fig. 5</a>. Bars represent the mean ± SEM of 3 independent biological replicates, each replicate analyzed in triplicate. (<i>C</i>) <i>SEP3</i> downregulation suppresses <i>gis5</i> curly leaf phenotype. Plants of the indicated genotypes were grown under LD at 23°C and photographed at flowering stages. Scale bar: 1 cm. (<i>D</i>) A model explaining the temperature-dependent effect of the <i>gis5</i> allele. The replication fork is represented and works normally at 18°C (green arrow) but is stalled at 24°C (red arrow). Small colored circles represent Polε (leading strand) and Polδ (lagging strand). At higher temperatures Polδ is delayed (red circle), triggering a DNA replication stress response and increased H3K4me3 at the <i>SEP3</i> locus which participates in a positive feedback loop with <i>FT</i> to induce flowering and the curly leaf phenotype. Blue arrows indicate tentative relationships, not tested yet. The Compass complex was tentatively included because of its role in H3K4me3 establishment. Orange short lines represent trimethylation in H3K4.</p

<i>FT</i> is required for <i>gis5</i> early flowering.

<p>(<i>A</i>-<i>B</i>) Loss of <i>FT</i> function suppresses most of <i>gis5</i> early flowering phenotype. Plants of the genotypes indicated on the abscissa were grown under LD (<i>A</i>) or SD (<i>B</i>) at 23°C and flowering recorded as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004975#pgen.1004975.g001" target="_blank">Fig. 1A</a>. Bars represent the mean ± SEM of at least 12 plants for each genotype. (<i>C</i>-<i>D</i>) The increase in <i>FT</i> expression is temperature-dependent in <i>gis5</i> mutants. WT and <i>gis5</i> mutant plants were grown for 10 days under continuous light at either 18°C or 24°C (<i>C</i>), or for 21 days under SD at either temperature. Plants grown under SD were harvested at the end of the photoperiod. Total RNA was extracted and <i>FT</i> transcripts quantitated by qRT-PCR relative to <i>UBQ10</i> mRNA. Bars represent the mean ±SEM of 3 independent biological replicates, each replicate analyzed in triplicate. (<i>E</i>) Phloem specific <i>FT</i> expression is required for <i>gis5</i> early flowering. <i>gis5</i> mutants were crossed to transgenic lines bearing artificial microRNAs against <i>FT</i> expressed under specific promoters (<i>FD</i> for apical meristem specific expression, <i>SUC2</i> for phloem specific expression and <i>35S</i> for high and constitutive expression [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004975#pgen.1004975.ref003" target="_blank">3</a>]. These genotypes and the corresponding controls (indicated on the abscissa) were grown under LD at 23°C and flowering recorded as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004975#pgen.1004975.g001" target="_blank">Fig. 1A</a>. Bars represent the mean ± SEM of at least 12 plants for each genotype.</p

<i>gis5</i> phenotypes are temperature-dependent.

<p>(<i>A</i>) <i>gis5</i> mutants flower early. WT, <i>gis5</i> and <i>gi-2</i> single mutants and <i>gis5 gi-2</i> double mutant lines were grown under LD at 23°C. The total leaf number (cauline and rosette leaves) was recorded at the time of flowering. Bars represent the mean ± SEM of at least 12 plants for each genotype. (<i>B</i> and <i>C</i>) <i>gis5</i> mutants display a temperature-dependent curly leaf phenotype. WT and <i>gis5</i> mutants were grown under LD at the indicated temperatures either on soil (<i>B</i>) or MS agar plates (<i>C</i>) and photographed at flowering. Scale bar: 1 cm. (<i>D</i> and <i>E</i>) <i>gis5</i> mutants display a temperature-dependent early flowering phenotype under SD. WT and <i>gis5</i> mutants were grown under LD (<i>D</i>) or SD (<i>E</i>) at the indicated temperatures (abscissas) and flowering time was recorded as in (<i>A</i>). Bars represent the mean ± SEM of at least 12 plants for each genotype.</p

The <i>gis5</i> allele of the catalytic subunit of DNA polymerase δ is thermosensitive.

<p>(<i>A</i>) The <i>gis5</i> mutants display a DNA replication stress response only at higher temperatures. WT and <i>gis5</i> mutant plants were grown for 10 days under continuous light at either 18°C or 24°C. Total RNA was extracted and <i>BRCA1</i> and <i>RAD51</i> transcripts quantitated by Reverse Transcriptase-PCR (qRT-PCR) relative to <i>UBQ10</i> mRNA In each panel, WT mRNA levels at 18°C were scaled to one. Bars represent the mean ±SEM of 3 independent biological replicates, each replicate analyzed in triplicate. (<i>B</i>) Homologous Recombination (HR) increases in <i>gis5</i> mutants in a temperature-dependent manner. The <i>gis5</i> mutant was crossed with HR reporter lines bearing either direct (1415) or inverted (1406) tandem repeats of a disrupted GUS gene [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004975#pgen.1004975.ref031" target="_blank">31</a>]. The repeats overlapp by 618 bp and a recombination event restores GUS activity. WT and <i>gis5</i> mutants bearing the reporter constructs were grown in LD at either 18°C or 24°C. Fully expanded 1^st pair-leaves were fixed, stained with X-Gluc and photographed. Dots (HR events) were quantified as described in Materials and Methods. (<i>C</i>) <i>gis5</i> mutant cells are delayed in the G2-M transition. A <i>PCycB1;1</i>:<i>GUS</i> reporter line [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004975#pgen.1004975.ref032" target="_blank">32</a>] was introduced into <i>gis5</i> mutants by crossing. Seven day old seedlings grown on LD and vertical MS agar plates at either 18 or 24°C were fixed and stained with X-gluc as described in Materials and Methods. (<i>D</i>) <i>gis5</i> mutants do not complete their life cycle at 28°C. WT (left) and <i>gis5</i> (right) mutants were grown on MS media at 28°C under LD and photographed at flowering.</p

The <i>SEP3</i> locus is enriched in H3K4me3 and H3Ac in a temperature dependent manner.

<p>WT and <i>gis5</i> mutant plants were grown for 10 days under continuous light at either 18°C or 24°C. Enrichment in H3K4me3, H3Ac and H3K27me3 was determined by ChIP followed by qRT-PCR of the fragments depicted in the top panel. Data was relativized to a UBQ10 fragment (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004975#pgen.1004975.s004" target="_blank">S4 Fig</a>. for data presented as a fraction of input). Bars represent the mean ± SEM of 5–6 independent biological replicates.</p

The expression of <i>SEP</i> genes increases in a temperature-dependent way in <i>gis5</i> mutants.

<p>(<i>A</i>-<i>B</i>) WT and <i>gis5</i> mutant plants were grown for 10 days under continuous light (<i>A</i>) at either 18°C or 24°C, or for 21 days under SD (<i>B</i>) at either temperature, together with <i>ft</i> and <i>gis5 ft</i> mutant lines. Plants grown under SD were harvested at the end of the light period. Total RNA was extracted and <i>SEP</i> transcripts quantitated by qRT-PCR relative to <i>UBQ10</i> mRNA. In each panel, WT mRNA levels at 18°C were scaled to one. Bars represent the mean ±SEM of 3 independent biological replicates, each replicate analyzed in triplicate.</p

<i>gis5</i> affects the catalytic subunit of Polδ.

<p>(<i>A</i>) Positional cloning of <i>gis5</i>. Representation of a chromosome V interval, overlapping BACs and markers (arrows) used to screen for recombinants. The <i>gis5</i> interval is flanked by markers CER436434 and CER436454. A C>T transition was detected in the 18^th exon of the At5g63960 <i>locus</i> (<i>POLD1</i>) leading to an Ala to Val substitution in the catalytic subunit of Polδ. (<i>B</i>) The WT <i>POLD1</i> sequence complements <i>gis5</i> flowering phenotype. Four independent <i>gis5</i> transgenic lines (G1 to G4), bearing a WT fragment of <i>POLD1</i>, were grown under SD (left panel) or LD (right panel) conditions. Bars represent the mean ± SEM of at least 12 plants for each genotype. (<i>C</i>) Superposition of the structural model of the catalytic subunit of Polδ and the yeast pol3 (pdb code: 3IAY). The program “Modeller” Version 9.13 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004975#pgen.1004975.ref062" target="_blank">62</a>] was used to construct the model using the X-ray structure of pol3 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004975#pgen.1004975.ref027" target="_blank">27</a>] (Top panel). Ribbon representation of the yeast Pol3, the Arabidopsis Polδ model and the DNA colored in green, gray and light orange respectively. Shown as sticks: ligand 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE (orange), 3IAY residues contacting the ligand (green). Shown as pink spheres are the Ca ions. Bottom panel: A detailed view of the modeled Polδ V707 (red sticks), which shows that the lateral chain points in the opposite direction of the substrate binding pocket.</p