Chromosome dynamics of the early meiotic cell cycle in S. cerevisiae

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.Includes bibliographical references.In every cell cycle the genetic material must be duplicated and transmitted to the daughter cells. Meiosis is a developmental program that allows a diploid cell to produce haploid progeny. The reduction in chromosome number obtained during meiosis requires specialized mechanisms that are absent during the canonical mitotic cell cycle. Although previous studies found strong similarities between pre-mitotic and pre-meiotic DNA replication, pre-meiotic S phase is longer than pre-mitotic S phase, suggesting that meiosis-specific events regulate the rate of DNA replication. Additionally, after DNA replication, homologous recombination is initiated by the introduction of hundreds DNA double-strand breaks (DSBs) into the genome to produce physical DNA exchanges, or crossovers, between homologous chromosomes. To investigate the chromosome dynamics of the early meiotic cell cycle, I performed comprehensive analysis of pre-meiotic DNA replication and DSB formation in budding yeast. Genome-wide studies of pre-meiotic DNA replication confirmed that the same replication origins are selected and activated in pre-meiotic and pre-mitotic cells, although replication was delayed at a large number of origins. These results indicate that the regulation of DNA replication is similar in the meiotic and mitotic cell cycles, but that the replication-timing program differs. Elimination of meiosis-specific cohesion or homologous recombination had no effect on the number or identity of early pre-meiotic origins. Analysis of cells sporulated in the presence of the replication inhibitor HU revealed a Cln3-dependent inhibition of meiotic entry. To map the locations of meiotic DSBs, I developed a method to detect meiotic ssDNA. Examination of the sites of ssDNA enrichment indicated that DSBs occur mainly in the promoters of active genes, consistent with previous studies of individual DSB sites.(cont.) Global analysis of the most common DSB sites revealed a non-random distribution of DSB "hotspots." In particular, DSB hotspots are over-enriched close to chromosome ends, which could explain why small chromosomes have a higher DSB density than large chromosomes. This mechanism could help ensure that all homologous chromosomes receive at least one crossover and segregate properly in meiosis. These studies also indicated that suppression of recombination at telomeres, centromeres and around the rDNA occurs by 3 distinct mechanisms.by Hannah G. Blitzblau.Ph.D

Production of 10-methyl branched fatty acids in yeast

Background: Despite the environmental value of biobased lubricants, they account for less than 2% of global lubricant use due to poor thermo-oxidative stability arising from the presence of unsaturated double bonds. Methyl branched fatty acids (BFAs), particularly those with branching near the acyl-chain mid-point, are a high-performance alternative to existing vegetable oils because of their low melting temperature and full saturation. Results: We cloned and characterized two pathways to produce 10-methyl BFAs isolated from actinomycetes and γ-proteobacteria. In the two-step bfa pathway of actinomycetes, BfaB methylates Δ9 unsaturated fatty acids to form 10-methylene BFAs, and subsequently, BfaA reduces the double bond to produce a fully saturated 10-methyl branched fatty acid. A BfaA-B fusion enzyme increased the conversion efficiency of 10-methyl BFAs. The ten-methyl palmitate production (tmp) pathway of γ-proteobacteria produces a 10-methylene intermediate, but the TmpA putative reductase was not active in E. coli or yeast. Comparison of BfaB and TmpB activities revealed a range of substrate specificities from C14-C20 fatty acids unsaturated at the Δ9, Δ10 or Δ11 position. We demonstrated efficient production of 10-methylene and 10-methyl BFAs in S. cerevisiae by secretion of free fatty acids and in Y. lipolytica as triacylglycerides, which accumulated to levels more than 35% of total cellular fatty acids. Conclusions: We report here the characterization of a set of enzymes that can produce position-specific methylene and methyl branched fatty acids. Yeast expression of bfa enzymes can provide a platform for the large-scale production of branched fatty acids suitable for industrial and consumer applications

Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions

During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLγ complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4Eme1. Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastroph

Separation of DNA Replication from the Assembly of Break-Competent Meiotic Chromosomes

The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not strictly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms

RNA Methylation by the MIS Complex Regulates a Cell Fate Decision in Yeast

For the yeast Saccharomyces cerevisiae, nutrient limitation is a key developmental signal causing diploid cells to switch from yeast-form budding to either foraging pseudohyphal (PH) growth or meiosis and sporulation. Prolonged starvation leads to lineage restriction, such that cells exiting meiotic prophase are committed to complete sporulation even if nutrients are restored. Here, we have identified an earlier commitment point in the starvation program. After this point, cells, returned to nutrient-rich medium, entered a form of synchronous PH development that was morphologically and genetically indistinguishable from starvation-induced PH growth. We show that lineage restriction during this time was, in part, dependent on the mRNA methyltransferase activity of Ime4, which played separable roles in meiotic induction and suppression of the PH program. Normal levels of meiotic mRNA methylation required the catalytic domain of Ime4, as well as two meiotic proteins, Mum2 and Slz1, which interacted and co-immunoprecipitated with Ime4. This MIS complex (Mum2, Ime4, and Slz1) functioned in both starvation pathways. Together, our results support the notion that the yeast starvation response is an extended process that progressively restricts cell fate and reveal a broad role of post-transcriptional RNA methylation in these decisions

RTG from starvation results in three distinct cell morphologies.

<p>(A) Representative morphologies of daughter cells following RTG from SPO throughout a meiotic time course. Top left panel: RTG at 0 hours, <i>i.e.</i>, after growth in BYTA, (RTG₀); top right panel: RTG at 3 hours (RTG₃), which is comparable to PH cells from solid nitrogen medium (bottom left panel). Bottom right panel: RTG at 6 hours (RTG₆). RTG₀ and RTG₃ cells were photographed after one complete cell cycle, corresponding to 160 minutes and 220 minutes after shift to rich medium, respectively (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002732#pgen.1002732.s001" target="_blank">Figure S1A</a>). RTG₆ cells were photographed three hours after shift to rich medium, at which point the majority of cells (>95%) had formed spores. Arrows indicate primary daughter cells upon RTG₀ and RTG₃. B) Quantification of axial ratio in wild-type (SAy821) cells upon RTG throughout a meiotic time course (red bars, left axis; n = 200 cells/time point) relative to percent of cells undergoing pre-meiotic DNA synthesis (<i>i.e.</i>, 4C cells) (blue diamonds, right axis, quantified by FACS, 3×10⁴ cells/time point) and percent cells undergoing meiotic divisions as assayed by DAPI staining (green triangles, right axis). Schematic at top defines axial ratio. The majority (>80%) of RTG₅, RTG₆, RTG₇, and RTG₉ cells either formed spores or remained unbudded three hours after shift to rich medium; axial ratio was therefore not quantified for these time points. C) <i>MAT </i><b>a</b>/α diploids (SAy821) (top left panel) or <i>MAT</i> α haploids (H224) (top right panel) were returned to growth in rich medium after meiotic induction. Arrows indicate primary buds. Bottom panels represent colony morphologies after growth on SLAD for 6 days. D) Distribution of axial ratios of primary daughter cells upon RTG₃ for strains in (C): wild-type diploid (SAy821 top panel), haploid <i>MAT</i> α (H224 bottom panel) (n = 200 cells/strain). RTG₀ is represented in red bars, RTG₃ in blue bars. E) Wild-type (SAy821), <i>flo11</i>Δ/Δ (SAy789) and <i>flo8</i>Δ/Δ (SAy905) daughter cell morphologies upon RTG₃ (top panels). Arrows indicate primary daughter cells. The same strains were photographed after growth on SLAD for 6 days (bottom panels). Axial ratios are quantified in (F) (n = 200 cells/strain).</p

Meiotic DNA replication profiles.

<p>(A) <i>Ime2-as1-myc</i> homozygous diploid cells (KBY518) were synchronized in meiS. DNA samples were collected every 7.5 minutes. Resulting samples were pooled and co-hybridized with a G1 DNA sample to a tiled genomic microarray. (B) Replication profiles for meiS (KBY518, red line), mitS (SB1505, blue line) and control G1 vs. G1 (SB1505, grey line) hybridizations were created by plotting the smoothed log₂ ratio (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002643#s4" target="_blank">Materials and Methods</a>) versus chromosome VII position. Triangles indicate the positions of Mcm2-7 binding sites prior to meiosis (red) and mitosis (blue). (C) The distribution of relative replication time for all origins (colored lines) and for the entire genome (black lines) is plotted for meiS (left panel) and mitS phase (right panel). (D) The replication time in meiS of Mcm2-7 binding sites that were present in both cell cycles were plotted as a function of mitS replication time. Assuming meiS is twice as long as mitS, the orange dashed line indicates the predicted meiS replication time if origins replicated with the same kinetics in meiS and mitS. The blue dashed line is the predicted replication time trend line if scaling were linear with respect to S phase length. The purple solid line is the second order polynomial best-fit model.</p

No relationship between DNA replication timing and recombination sites.

<p>(A) Chromosome VII replication profiles for pre-meiotic cells in the presence of 20 mM HU are shown for wild-type (H574, red), <i>rec8Δ</i> (H5187, orange) and <i>spo11Δ</i> (H5184, green) cells. Inverted triangles indicate the position of origins that are considered replicated in each strain. (B) Venn diagram summary of the experiment shown in (A), with the same color coding. (C) The distributions of replication timing in meiS are shown for the entire genome (black line), DSBs hotspots mapped by Spo11-oligo recovery (gray line), ssDNA enrichment in a <i>dmc1Δ</i> strain (brown line) and Spo11 binding in <i>rad50S</i> cells (green line). (D) The distributions of replication timing in meiS are shown for the entire genome (black line) and for axis association sites (blue line).</p

Centromeres replicate early in S phase.

<p>(A) The average expression level of origin proximal genes is plotted versus the time of replication in meiS. The red dotted lines indicate the population average. (B) The expression level distributions for meiS (left) and mitS (right) are plotted for the genes surrounding each origin for meiS early origins (red boxes) and mitS-only early origins (blue boxes). (C) The replication time for each centromere is indicated as a gray vertical bar compared to the distribution of replication time for the whole genome (black line) in meiS (left panel) and mitS (right panel). The mean replication time of the genome is indicated by the black dotted lines for each panel. (D) The replication time of each origin is plotted as a function of the distance of the origin from the closest centromere. MeiS early origins are indicated in red, mitS-only early origins are indicated in blue and late origins are colored black. (E) The data from (D) are summarized as box and whisker plots, with significance of the difference between mei-S and mitS-only early origins indicated.</p