SsDNA in S phase compared to G1 phase at different timepoints
ABSTRACT: To identify origins of replication in Lachancea waltii by mapping regions of ssDNA that are enriched in S phase. L. waltii cells are grown in the presence of hydroxyurea, which slows replication forks and causes the accumulation of ssDNA at the replication fork.
Project description:Array data detailing the progression of DNA replication in the yeast Lachancea waltii. L. waltii cells were pregrown in heavy isotope medium and synchronized at early S phase. They were then released into normal medium, wherein DNA replication proceeds. Replicated DNA molecules are thus of heavy-light (HL) composition as compared to unreplicated molecules, which are heavy-heavy (HH). 4 time points were taken and the percent of heavy-light DNA was determined at each time point. The heavy-heavy and heavy-light DNA molecules were separated by ultracentrifugation, differentially labeled, and hybridized to a genomic array for L. waltii. The array thus shows the progression of DNA replication.
Project description:DSBs were mapped genome-wide by ssDNA enrichment in cdc6-mn replication comprimsied strains. We mapped DSB sites by detecting DSB-associated ssDNA enrichment on microarrays. To test the role of DNA replication in DSB formation, we mapped ssDNA in a cdc6-mn replication depleted strain. ssDNA was isolated from cells after 5 hours in sporulation medium. As a reference, ssDNA isolated from cells at 0 hrs in sporulation medium prior to DSB formation was differentially labeled and co-hybridized to the same array. For each experiment, we have submitted biological replicates that were hybridized to separate arrays. For each experiment a dye swap was performed and shown to have no effect on the data observed, although not all experiments in this series include the dye swap sample (we have included only two representative experiments for each strain).
Project description: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 directly 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. Multiple studies of meiotic chromosomes were undertaken. To study DNA replication, the locations of replicative helicase (Mcm2-7) were mapped in pre-meiotic and pre-mitotic cells, and DNA replication profiles were created for pre-meiotic S (meiS) and pre-mitotic S (mitS) phases. Early origins were mapped in hydroxyurea for wild-type cells in mitS + 200mM HU, and meiS +20mM HU for wild-type, sml1, rec8 and spo11 deletion cells. Rec8, Hop1 and Red1 binding to meiotic chromosomes was evaluated using ChIP-chip in wild-type cells with and without 20 mM HU, and in cdc6-mn and clb5 clb6 delete cells. Finally, meiotic DNA double-strand breaks (DSBs) were mapped in cdc6-mn dmc1 delete cells by measuring the ssDNA that accumulates at DSB hotspots. This SuperSeries is composed of the following subset Series: GSE35658: Chromatin IP for Mcm2-7, Rec8, Hop1 and Red1 GSE35662: S phase and HU profiles in wild-type and mutant cells GSE35666: DSB formation in replication compromised cells
Project description:Meiotic DNA double stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. Although DSBs can be directly mapped using ChIP-Seq and antibody against ssDNA-associated proteins, genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method, single-stranded DNA (ssDNA) sequencing (SSDS), that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS consists of a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. When applied to mapping meiotic DSBs, the use of SSDS reduces the non-specific dsDNA background more than ten-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired. Development and validation of the method, SSDS, for the specific detection of ssDNA-derived and dsDNA-derived fragments in sequencing libraries and enrichment of ssDNA-derived fragments. SSDS was used to detect meiotic DSBs in 9R/13R mice.
Project description:ssDNA enrichment was used to map and compare DSB hotspots in dmc1, pch2 dmc1, sir2 dmc1, orc1-161 dmc1, dmc1 rdnadelete and dmc1 chr2:12 translocation strains. 6 samples, 2 replicates each. Averaged data is available as a supplementary file on the Series record (below).
Project description:We have developed a method to analyze single-stranded DNA (ssDNA) formation on a genomic scale by using microarrays. Using this technique we have assessed the location and the amount of ssDNA in S. cerevisiae during DNA replication. We have observed that when replication is impeded by hydroxyurea, ssDNA formation can be detected in both wild type and the checkpoint-deficient rad53 cells. However, while wild type cells showed ssDNA formation at only a subset of origins, rad53 cells formed ssDNA at virtually all known origins. Moreover, in rad53 cells the ssDNA regions did not expand over time, presumably due to collapsed replication forks. We also applied this method to map origins in S. pombe, taking advantage of the conserved replication checkpoint function by Cds1, the homolog of Rad53 in S. pombe. Keywords: ssDNA, HU, replication, time course Overall design: In order to investigate the dynamics of ssDNA formation on a genomic scale, we harvested cells at discrete times after releasing them from late G1 phase arrest with alpha factor into a synchronous S phase in the presence of 200 mM HU. Chromosomal DNA isolated from these S phase samples and an alpha factor arrested G1 control sample were differentially labeled with Cy-conjugated deoxyribonucleotides by random priming and synthesis without denaturation of the DNA, followed by co-hybridization to a microarray. Because the labeling was done without denaturation of the template DNA, single-stranded regions of the genome should preferentially act as templates for dye incorporation. Comparison of experimental (S phase) and control (G1 phase) samples from the microarray hybridization revealed regions of the genome that became single-stranded in S phase. We also assessed the total percentage of ssDNA in the samples by blotting native (undenatured) genomic DNA and fully denatured genomic DNA, followed by hybridization with a genomic DNA probe. The calculated total percentages of ssDNA in the samples were then used to normalize the raw ratio of ssDNA (S/G1) (raw data) to generate the normalized ratio of ssDNA (S/G1) (raw normalized data). The normalized relative ratio of ssDNA was then smoothed over a 4 kb window (smoothed data) via Fourier transformation. For origin mapping in S. pombe, we used S. pombe wild type and deltacds1 (cds1 encodes for the homolog of Rad53) cells in a comparative analysis. DNA isolated from cells that were exposed to HU (“early S phase” sample) and cells that were starved for nitrogen source (G1 phase control sample) were differentially labeled with Cy-conjugated dUTPs without denaturation of the template DNA to enrich for labeling of ssDNA region in the genome. These DNAs were then co-hybridized to a microarray. The relative amount of ssDNA was quantitated as the ratio of fluorescent signal from the “early S phase” sample to that from the control sample. The raw ratio of ssDNA (early S/G1) was then normalized by the total percentage of ssDNA in the samples similarly as for S. cerevisiae data. The raw normalized ratio was then smoothed over a 12 kb window via Fourier transformation.
Project description:DNA double strand breaks (DSBs) in repetitive sequences are a potent source of genomic instability, due to the possibility of non-allelic homologous recombination (NAHR). Repetitive sequences are especially at risk during meiosis, when numerous programmed DSBs are introduced into the genome to initiate meiotic recombination 1. Within the budding yeast repetitive ribosomal (r)DNA array, meiotic DSB formation is prevented in part through Sir2-dependent heterochromatin 2,3. Here, we demonstrate that the edges of the rDNA array are exceptionally susceptible to meiotic DSBs, revealing an inherent heterogeneity within the rDNA array. We find that this localised DSB susceptibility necessitates a border-specific protection system consisting of the meiotic ATPase Pch2 and the origin recognition complex subunit Orc1. Upon disruption of these factors, DSB formation and recombination specifically increased in the outermost rDNA repeats, leading to NAHR and rDNA instability. Strikingly, the Sir2-dependent heterochromatin of the rDNA itself was responsible for the induction of DSBs at the rDNA borders in pch2? cells. Thus, while Sir2 activity globally prevents meiotic DSBs within the rDNA, it creates a highly permissive environment for DSB formation at the heterochromatin/euchromatin junctions. Heterochromatinised repetitive DNA arrays are abundantly present in most eukaryotic genomes. Our data define the borders of such chromatin domains as distinct high-risk regions for meiotic NAHR, whose protection may be a universal requirement to prevent meiotic genome rearrangements associated with genomic diseases and birth defects. This SuperSeries is composed of the following subset Series: GSE30071: ssDNA mapping in dmc1 strains GSE30072: ChIP-chip of DSB factors in wild type and pch2 strains Two types of study were undertaken to understand the regulation of meiotic DSB formation close to repetitive DNA elements in yeast. First, DSBs were mapped using ssDNA enrichment in strains isogenic for a dmc1 mutation, and also including pch2 delete, orc1-161, rdna delete and a reciprocal translocation between chromosomes 2 and 12 (trans2to12). Second, the association of the DSB factors Hop1, Rec114, Mer2, and Mre1, as well as total histone H3 and H3K4-trimethylation were measured by ChIP-chip analysis in wild-type and pch2 delete strains.
Project description:In response to DNA replication stress, DNA replication checkpoint is activated to maintain fork stability, a process critical for maintenance of genome stability. However, how DNA replication checkpoint regulates replication forks remain elusive. Here we show that Rad53, a highly conserved replication checkpoint kinase, functions to couple leading and lagging strand DNA synthesis. In wild type cells under HU induced replication stress, synthesis of lagging strand, which contains ssDNA gaps, is comparable to leading strand DNA. In contrast, synthesis of lagging strand is much more than leading strand, and consequently, leading template ssDNA coated with ssDNA binding protein RPA was detected in rad53-1 mutant cells, suggesting that synthesis of leading strand and lagging strand DNA is uncoupled. Mechanistically, we show that replicative helicase MCM and leading strand DNA polymerase Pole move beyond actual DNA synthesis and that an increase in dNTP pools largely suppresses the uncoupled leading and lagging strand DNA synthesis. Our studies reveal an unexpected mechanism whereby Rad53 regulates replication fork stability. Overall design: We synchronized yeast cells (Wile type and other mutant cells) at G1 and released into early S phase in the presence of BrdU, and hydroxyurea (HU). We then performed BrdU immunoprecipitation using anti-BrdU antibodies following single-strand DNA library preparation and sequencing. We also performed protein ChIP followed by single-strand DNA sequencing (ChIP-ssSeq) for MCM6 and Pol ɛ. The sequencing tag was mapped to both Watson (red) and Crick (blue) strands of the reference genome.
Project description:A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). To examine early replication dynamics on a genome-wide scale in orc4Y232C cells, we utilized an assay that was previously developed in our lab. This assay uses microarray hybridization to measure the levels of single stranded DNA exposed at replication forks. While we do not fully understand the molecular processes that give rise to peaks of different amplitudes—e.g., number of cells that have activated a particular origin vs. amount of ssDNA revealed at different forks—the results from different replicates of the experiment are highly reproducible. We find that origins that are known to fire early and are efficient produce the peaks of greatest magnitude, while later firing and less efficient origins produce smaller or no peaks in this assay. The characteristic time and/or efficiency of origin firing within the S phase is altered for at least 15% of the 300 yeast origins. Among the origins with delayed/reduced origin firing are normally early-firing origins adjacent to centromeres. Overall design: To investigate the dynamics of ssDNA formation on a genomic scale, we harvested cells at discrete times after releasing them from late G1 phase arrest with alpha factor into a synchronous S phase in the presence of 200 mM HU. Chromosomal DNA isolated from these S phase samples and an alpha factor arrested G1 control sample were differentially labeled with Cy-conjugated deoxyribonucleotides by random priming and synthesis without denaturation of the DNA, followed by co-hybridization to a microarray. Because the labeling was done without denaturation of the template DNA, single-stranded regions of the genome should preferentially act as templates for dye incorporation. Comparison of experimental (S phase) and control (G1 phase) samples from the microarray hybridization revealed regions of the genome that became single-stranded in S phase. The calculated total percentages of ssDNA in the samples were then used to normalize the raw ratio of ssDNA (S/G1) (raw data) to generate the normalized ratio of ssDNA (S/G1) (raw normalized data). The normalized relative ratio of ssDNA was then smoothed over a 22 kb window (smoothed data) via loess smoothing. Peaks of S-phase specific ssDNA accumulation mark locations of active origins.
Project description:Mammalian DNA replication starts at distinct chromosomal sites in a tissue-specific pattern coordinated with transcription, but previous studies have not yet identified a chromatin modification that correlates with the initiation of DNA replication. This submission is associated with a paper in which we report that replication initiation events are associated with a high frequency of methylation of histone H3 on lysine K79 (H3K79Me2 and H3K79Me3). H3K79Me2-containing chromatin exhibited the highest enrichment of replication initiation events observed in a single chromatin modification. Importantly, H3K79 methylation was enriched in chromatin containing a replicator (a DNA sequence capable of initiating DNA replication), but not in chromatin containing a mutant replicator that could not initiate replication. The association of H3K79Me2 with replication initiation sites was independent and not synergistic with other chromatin modifications. H3K79 methylation exhibited a wider distribution and greater abundance during S-phase, but regions of chromatin that were only modified during S-phase were not enriched in replication initiation events. In addition, the paper shows that depletion of DOT1L, the sole enzyme responsible for H3K79 methylation, triggered limited genomic over-replication. These data are consistent with the hypothesis that methylation of H3K79 associates with replication origins and marks replicated chromatin during S-phase to prevent re-replication and preserve genomic stability. Evaluation of HeK79 methylation in chromatin samples from cell cycle fractionated K562 leukemia cells. Unsyncrhonized untreated cultures of K562 cells were fractionated by size using centrifugal elutriation. Chromatin was isolated and subject to ChIP-Seq with antibodies directed against dimethylated and trimethylated lysine on histone H3.