Ablating ATR in mouse meiosis and its consequences for synapsis, recombination and meiotic surveillance mechanisms

Meiosis is a fundamental part in the life cycle of sexual species. It denotes a specialised cell division that halves chromosome numbers to generate haploid gametes for reproduction. Cells unable to competently progress through meiotic prophase activate cell surveillance mechanisms causing their elimination. Given the importance of DNA damage kinases like ATR in facilitating mitotic cell surveillance mechanisms, I characterized Atr-deficient spermatocytes to determine the importance of ATR for mammalian meiosis. I found that ATR ensures efficient chromosome synapsis, and that that is partially independent of meiotic recombination. In addition, ATR has three distinct roles in meiotic recombination. Firstly, during nucleolytic processing, it acts to regulate SPO11-oligonucleotide size when ATM is deleted. Secondly, it is required for accurate RAD51 and DMC1 recruitment to DSBs. Thirdly, it regulates the timing of DNA DSB repair on both unsynapsed and synapsed chromosomes. Finally I found that the loss of ATR is unable to rescue meiotic arrest in multiple meiotic mutants, including mice deficient for the other DNA damage PIKKs ATM and DNA-PK. My findings reveal multiple roles for ATR in male mouse meiosis

Opening a new window to other worlds with spectropolarimetry

A high level of diversity has already been observed among the planets of our own Solar System. As such, one expects extrasolar planets to present a wide range of distinctive features, therefore the characterisation of Earth- and super Earth-like planets is becoming of key importance in scientific research. The SEARCH (Spectropolarimetric Exoplanet AtmospheRe CHaracerisation) mission proposal of this paper represents one possible approach to realising these objectives. The mission goals of SEARCH include the detailed characterisation of a wide variety of exoplanets, ranging from terrestrial planets to gas giants. More specifically, SEARCH will determine atmospheric properties such as cloud coverage, surface pressure and atmospheric composition, and may also be capable of identifying basic surface features. To resolve a planet with a semi major axis of down to 1.4AU and 30pc distant SEARCH will have a mirror system consisting of two segments, with elliptical rim, cut out of a parabolic mirror. This will yield an effective diameter of 9 meters along one axis. A phase mask coronagraph along with an integral spectrograph will be used to overcome the contrast ratio of star to planet light. Such a mission would provide invaluable data on the diversity present in extrasolar planetary systems and much more could be learned from the similarities and differences compared to our own Solar System. This would allow our theories of planetary formation, atmospheric accretion and evolution to be tested, and our understanding of regions such as the outer limit of the Habitable Zone to be further improved.Comment: 23 pages, accepted for publication in Experimental Astronom

Recombinogenic Conditions Influence Partner Choice in Spontaneous Mitotic Recombination

<div><p>Mammalian common fragile sites are loci of frequent chromosome breakage and putative recombination hotspots. Here, we utilized Replication Slow Zones (RSZs), a budding yeast homolog of the mammalian common fragile sites, to examine recombination activities at these loci. We found that rates of <i>URA3</i> inactivation of a <i>hisG-URA3-hisG</i> reporter at RSZ and non-RSZ loci were comparable under all conditions tested, including those that specifically promote chromosome breakage at RSZs (hydroxyurea [HU], <i>mec1Δ sml1Δ</i>, and high temperature), and those that suppress it (<i>sml1Δ</i> and <i>rrm3Δ</i>). These observations indicate that RSZs are not recombination hotspots and that chromosome fragility and recombination activity can be uncoupled. Results confirmed recombinogenic effects of HU, <i>mec1Δ sml1Δ</i>, and <i>rrm3Δ</i> and identified temperature as a regulator of mitotic recombination. We also found that these conditions altered the nature of recombination outcomes, leading to a significant increase in the frequency of <i>URA3</i> inactivation via loss of heterozygosity (LOH), the type of genetic alteration involved in cancer development. Further analyses revealed that the increase was likely due to down regulation of intrachromatid and intersister (IC/IS) bias in mitotic recombination, and that RSZs exhibited greater sensitivity to HU dependent loss of IC/IS bias than non RSZ loci. These observations suggest that recombinogenic conditions contribute to genome rearrangements not only by increasing the overall recombination activity, but also by altering the nature of recombination outcomes by their effects on recombination partner choice. Similarly, fragile sites may contribute to cancer more frequently than non-fragile loci due their enhanced sensitivity to certain conditions that down-regulate the IC/IS bias rather than intrinsically higher rates of recombination.</p></div

Rate of <i>URA3</i> inactivation in YPD and in HU.

<p><b>A, B.</b> Rate of <i>URA3</i> inactivation at the indicated locus in WT haploid- or diploid- strains grown in YPD (A) or in 10 mM HU (B). 95% Confidence Limits (CLs) for each value were calculated as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931-Wierdl1" target="_blank">[47]</a>. For each locus, rate measurements from two independently derived strains were obtained by the methods of median (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931.s002" target="_blank">Figure S2</a>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931-Lea1" target="_blank">[24]</a>. <b>C.</b> The average recombination rate (and 95% CLs) of the haploid- and diploid- strains analyzed in <b>A</b> and <b>B.</b> “HU/YPD”: The effects of HU over YPD on recombination rate was expressed as the ratio between the two average rates. <b>D, E.</b> Effects of HU on rate of <i>URA3</i> inactivation at each locus. Graphic representation of the data presented in <b>A</b> and <b>B.</b> Black and grey circles correspond to rate measurements in HU and YPD, respectively. Capped lines indicate 95% CLs. The red number at the top of each box is the ratio between the average rate values in HU and YPD at the indicated locus; the extent increase conferred by HU was statistically significant at every locus (Chi square test, p<0.05).</p

<i>sml1Δ</i> regulation of mitotic recombination.

<p><b>A, B.</b> Rates of <i>URA3</i> inactivation at the indicated locus in <i>sml1Δ</i> haploid and diploid strains grown in YPD. 95% Confidence Limits (CLs) for each value were calculated as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931-Wierdl1" target="_blank">[47]</a>. For each locus except for ORI, rate measurements from two independently derived strains were obtained by methods of median (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931.s002" target="_blank">Figure S2</a>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931-Lea1" target="_blank">[24]</a>. <b>C, D.</b> Effects of <i>sml1Δ</i> on rate of <i>URA3</i> inactivation. Black and grey circles correspond to rate measurements in <i>sml1Δ</i> (panels <b>A</b> and <b>B</b>) and WT (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen-1003931-g002" target="_blank">Figure 2A</a>), respectively. Capped lines indicate 95% CLs. The number at the top of each box is the ratio between average rate values in <i>sml1Δ</i> and WT at each locus. * denotes a statistically significant change (Chi square test, p<0.05). Numbers in red and blue denote statistically significant increase or decrease, respectively. <b>E.</b> Locus specific effects of <i>sml1Δ</i> on rate of <i>URA3</i> inactivation. ^a The average rate at the indicated locus in <i>sml1Δ</i> was normalized to the corresponding value in WT (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen-1003931-g002" target="_blank">Figure 2</a>). Numbers in red and blue denote statistically significant increase or decrease, respectively (Chi square test, p<0.05). NSS: Not Statistically Significant. <b>F.</b> Effect of <i>sml1Δ</i> on IC/IS bias. Fraction of 5FOA^R colonies that had undergone <i>URA3</i> inactivation via an IC/IS mediated event ^a The fraction in <i>sml1Δ</i> at each locus was normalized to the corresponding value in WT (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen-1003931-g003" target="_blank">Figure 3E</a>). ^b Statistical analysis (Fischer's exact test) was performed on the effects of <i>sml1Δ</i>. The number in red denotes statistically significant increase.</p

Experimental system.

<p><b>A.</b> Recombination activity at five different loci in chromosome III (ChrIII) was assessed by monitoring <i>URA3</i> inactivation in <i>hisG-URA3-hisG</i> reporter introduced at each of the five indicated site. The “kb from TEL” and “kb from CEN” are distances from the left telomere and the centromere (CEN) in kb, respectively. Grey boxes represent the six RSZs referred to as I through IV in ChrIII <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931-Cha1" target="_blank">[3]</a>. T: replication fork termination site; Open circle: active replication origin; Grey circle: tRNA gene; Triangle: Ty element. <b>B.</b> Summary of notable features at each locus examined. <b>C.</b> Mechanisms of <i>URA3</i> inactivation. Heterozygous diploid strains carrying a single copy of <i>hisG-URA3-hisG</i> (blue circle; <i>URA+</i>) were grown under specified conditions and selected for 5FOA resistance. “Molecular events” summarize possible mechanisms of <i>URA3</i> inactivation. Co-segregation of <i>ura3</i> chromatids at mitosis would lead to a 5FOA^R colony (pink circle) that no longer carries the <i>hisG-URA3-hisG</i> allele. The resulting diploid would carry either the “pop-out” and a single copy of WT (i, ii), two copies of WT (iii, iv), or just one copy of WT (v, vi). L and R; the immediate upstream and downstream sequences of each insertion locus utilized for targeted introduction of the tester construct via homologous recombination (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931.s001" target="_blank">Figure S1</a>). INT: the allele containing the tester construct.</p

Design criteria for experiments to measure the breakdown voltage of insulating gases in uniform electric fields

Round robin breakdown experiments in (quasi)uniform electric fields have been performed in 7 different laboratories for air, HFO1234ze(E)/N2 (20 %/80 %), and SF6 as example gases. The experiments are supported and discussed with an extended literature review of gas-physical phenomena and constructional setup parameters influencing the results. The goals are to investigate a) the influence of certain experimental parameters on the outcome and scatter of breakdown measurements and b) to define suitable and practical setups for breakdown experiments and evaluation methods for the main experiments of Cigre working group D1.67 investigating new non-SF6 insulating gases and gas mixtures. The final recommendation is to use polished plane-plane Rogowski electrodes with a spacing of 5 mm in a self-supporting PEEK frame, and an AC voltage rate of rise around 0.1 kV/s. The value of this study goes beyond the work of the working group D1.67 and serves as basic recommendation for all groups performing breakdown experiments in gaseous insulation systems

ATR is a multifunctional regulator of male mouse meiosis

Meiotic cells undergo genetic exchange between homologs through programmed DNA double-strand break (DSB) formation, recombination and synapsis. In mice, the DNA damage-regulated phosphatidylinositol-3-kinase-like kinase (PIKK) ATM regulates all of these processes. However, the meiotic functions of the PIKK ATR have remained elusive, because germline-specific depletion of this kinase is challenging. Here we uncover roles for ATR in male mouse prophase I progression. ATR deletion causes chromosome axis fragmentation and germ cell elimination at mid pachynema. This elimination cannot be rescued by deletion of ATM and the third DNA damage-regulated PIKK, PRKDC, consistent with the existence of a PIKK-independent surveillance mechanism in the mammalian germline. ATR is required for synapsis, in a manner genetically dissociable from DSB formation. ATR also regulates loading of recombinases RAD51 and DMC1 to DSBs and recombination focus dynamics on synapsed and asynapsed chromosomes. Our studies reveal ATR as a critical regulator of mouse meiosis

ATR is a multifunctional regulator of male mouse meiosis

Meiotic cells undergo genetic exchange between homologs through programmed DNA double-strand break (DSB) formation, recombination and synapsis. In mice, the DNA damage-regulated phosphatidylinositol-3-kinase-like kinase (PIKK) ATM regulates all of these processes. However, the meiotic functions of the PIKK ATR have remained elusive, because germline-specific depletion of this kinase is challenging. Here we uncover roles for ATR in male mouse prophase I progression. ATR deletion causes chromosome axis fragmentation and germ cell elimination at mid pachynema. This elimination cannot be rescued by deletion of ATM and the third DNA damage-regulated PIKK, PRKDC, consistent with the existence of a PIKK-independent surveillance mechanism in the mammalian germline. ATR is required for synapsis, in a manner genetically dissociable from DSB formation. ATR also regulates loading of recombinases RAD51 and DMC1 to DSBs and recombination focus dynamics on synapsed and asynapsed chromosomes. Our studies reveal ATR as a critical regulator of mouse meiosis