Extraction of genomic DNA from yeasts for PCR-based applications

We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)–SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 107 cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤3500 bp

Rpb9-deficient cells are defective in DNA damage response and require histone H3 acetylation for survival

Rpb9 is a non-essential subunit of RNA polymerase II that is involved in DNA transcription and repair. In budding yeast, deletion of RPB9 causes several phenotypes such as slow growth and temperature sensitivity. We found that simultaneous mutation of multiple N-terminal lysines within histone H3 was lethal in rpb9Δ cells. Our results indicate that hypoacetylation of H3 leads to inefficient repair of DNA double-strand breaks, while activation of the DNA damage checkpoint regulators γH2A and Rad53 is suppressed in Rpb9-deficient cells. Combination of H3 hypoacetylation with the loss of Rpb9 leads to genomic instability, aberrant segregation of chromosomes in mitosis, and eventually to cell death. These results indicate that H3 acetylation becomes essential for efficient DNA repair and cell survival if a DNA damage checkpoint is defective

Acetylation of H3 K56 Is Required for RNA Polymerase II Transcript Elongation through Heterochromatin in Yeast▿

In Saccharomyces cerevisiae SIR proteins mediate transcriptional silencing, forming heterochromatin structures at repressed loci. Although recruitment of transcription initiation factors can occur even to promoters packed in heterochromatin, it is unclear whether heterochromatin inhibits RNA polymerase II (RNAPII) transcript elongation. To clarify this issue, we recruited SIR proteins to the coding region of an inducible gene and characterized the effects of the heterochromatic structure on transcription. Surprisingly, RNAPII is fully competent for transcription initiation and elongation at the locus, leading to significant loss of heterochromatin proteins from the region. A search for auxiliary factors required for transcript elongation through the heterochromatic locus revealed that two proteins involved in histone H3 lysine 56 acetylation, Rtt109 and Asf1, are needed for efficient transcript elongation by RNAPII. The efficiency of transcription through heterochromatin is also impaired in a strain carrying the K56R mutation in histone H3. Our results show that H3 K56 modification is required for efficient transcription of heterochromatic locus by RNAPII, and we propose that transcription-coupled incorporation of H3 acetylated K56 (acK56) into chromatin is needed for efficient opening of heterochromatic loci for transcription

Alterations of distance between the Fkh1/2 binding sites in <i>ARS607</i>.

<p><b>(A)</b> Schematic representation of <i>ARS607</i> origins inserted into the <i>GAL</i>-<i>VPS13</i> locus. Approximate locations of Fkh1/2 consensus binding sites (blue boxes) and the ACS (pink box) are indicated. In all constructs except wt <i>ARS607</i> the ACS-distal (3’) Fkh1/2 binding site was mutated (red X) and a new Fkh1/2 site was introduced at various distances from the ACS-proximal (5’) site. <b>(B)</b> Depiction of modified <i>ARS607</i> origins with small insertions and deletions between Fkh1/2 binding sites. Approximate locations of Fkh1/2 consensus binding sites (blue boxes), the ACS (pink box), poly-A track and sites of nucleotide insertions or deletions are indicated. Detailed sequences of all modified origin loci are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006588#pgen.1006588.s004" target="_blank">S4 Fig</a>. <b>(C)</b> Fkh1 binding to <i>ARS607</i> with altered distances between Fkh1/2 sites as determined by ChIP assay. Fkh1 occupancy at mutant <i>ARS607</i> is shown relative to its binding to the native <i>ARS607</i> locus in the same strain. A strain with no ARS in the <i>VPS13</i> locus and the native late-replicating origin <i>ARS522</i> that contains no Fkh1/2 binding sites are shown as controls (<i>VPS13</i> and <i>ARS522</i>, respectively). <b>(D)</b> Relative copy number of <i>VPS13</i>-<i>ARS607</i> DNA in HU-arrested cells. Cells were arrested in G1 with α-factor and then released into HU-containing media for 45 and 75 minutes. Graphs show the ratio of <i>VPS13</i>-<i>ARS607</i> and late-replicating <i>ARS522</i> loci, the ratio in G1-arrested cells was set as 1. Relative copy number of the native <i>ARS607</i> locus is shown as control. Full data for each strain is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006588#pgen.1006588.s001" target="_blank">S1 Fig</a>. <b>(E)</b> Mcm4 binding to <i>ARS607</i> loci with altered distances between Fkh1/2 sites as determined by ChIP assay. Mcm4 occupancy at mutant <i>ARS607</i> is shown relative to its binding to the native <i>ARS607</i> origin in the same strain. A strain with no ARS in the <i>VPS13</i> locus and the native origin <i>ARS522</i> are shown as controls (<i>VPS13</i> and <i>ARS522</i>, respectively).</p

Recruitment of Fkh1 to replication origins requires precisely positioned Fkh1/2 binding sites and concurrent assembly of the pre-replicative complex

<div><p>In budding yeast, activation of many DNA replication origins is regulated by their chromatin environment, whereas others fire in early S phase regardless of their chromosomal location. Several location-independent origins contain at least two divergently oriented binding sites for Forkhead (Fkh) transcription factors in close proximity to their ARS consensus sequence. To explore whether recruitment of Forkhead proteins to replication origins is dependent on the spatial arrangement of Fkh1/2 binding sites, we changed the spacing and orientation of the sites in early replication origins <i>ARS305</i> and <i>ARS607</i>. We followed recruitment of the Fkh1 protein to origins by chromatin immunoprecipitation and tested the ability of these origins to fire in early S phase. Our results demonstrate that precise spatial and directional arrangement of Fkh1/2 sites is crucial for efficient binding of the Fkh1 protein and for early firing of the origins. We also show that recruitment of Fkh1 to the origins depends on formation of the pre-replicative complex (pre-RC) and loading of the Mcm2-7 helicase, indicating that the origins are regulated by cooperative action of Fkh1 and the pre-RC. These results reveal that DNA binding of Forkhead factors does not depend merely on the presence of its binding sites but on their precise arrangement and is strongly influenced by other protein complexes in the vicinity.</p></div

Analysis of Fkh1/2 binding sites at replication origins.

<p><b>(A)</b> Double Fkh1/2 binding sites (RYMAAYA) with different spacing and orientation were located throughout the yeast genome. The sites were then mapped onto a genome-wide early DNA replication initiation profile [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006588#pgen.1006588.ref008" target="_blank">8</a>]. Percentage of double Fkh1/2 sites that co-localize with early-replicating loci is shown. Black dashed line indicates the background and is calculated as average frequency of overlap between scrambled Fkh1/2 double consensus sites (in all orientations; with 50–100 bp gap) and early origins. <b>(B)</b> Distribution of double Fkh1/2 sites at late replication origins was analysed as in <b>A</b>. <b>(C)</b> The general pattern of sequence elements in early replication origins with divergent Fkh1/2 binding sites. Two Fkh1/2 sites (blue arrows) are separated by a stretch of 71–79 bp linker DNA that contains a poly-A track. Location of the ACS (pink ellipse) varies, but is typically found in close proximity to or overlapping with one Fkh1/2 site. The ACS-proximal Fkh1/2 consensus sequence is located on the DNA strand complementary to that containing the ACS. The model is based on alignment of 20 early origins containing divergent Fkh1/2 binding sites (see text and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006588#pgen.1006588.s003" target="_blank">S3 Fig</a> for details).</p

Alterations of Fkh1/2 site orientations in <i>ARS305</i>.

<p><b>(A)</b> Schematic representation of <i>ARS305</i> origins inserted into the <i>GAL</i>-<i>VPS13</i> locus. Approximate locations of Fkh1/2 consensus binding sites (blue arrows) and the ACS (pink box) are indicated. In 5’mut construct, the ACS-proximal Fkh1/2 site was disrupted by mutation (red X). In Fkh1/2 site reversal mutants, the sites were inverted relative to their original orientations (red arrows). <b>(B)</b> Fkh1 binding to <i>ARS305</i> loci with various orientations of Fkh1/2 sites as determined by ChIP assay. Fkh1 occupancy to <i>ARS305</i> mutants is shown relative to its binding to the native <i>ARS305</i> locus. Fkh1 binding to the native late-replicating origin <i>ARS522</i> is shown as control (<i>ARS522</i>). <b>(C)</b> Relative copy number of <i>VPS13</i>-<i>ARS305</i> DNA in HU-arrested cells. Cells were arrested in G1 with α-factor and then released into HU-containing media for 45 and 75 minutes. Graphs show the ratio of <i>VPS13</i>-<i>ARS305</i> and late-replicating <i>ARS522</i> loci, the ratio in G1-arrested cells was set as 1. Relative copy number of the native <i>ARS305</i> and origin-free <i>VPS13</i> loci are shown as controls. Full data for each strain is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006588#pgen.1006588.s002" target="_blank">S2 Fig</a>. <b>(D)</b> Mcm4 binding to <i>ARS305</i> loci with various orientations of Fkh1/2 sites as determined by ChIP assay. Mcm4 occupancy to mutant <i>ARS305</i> is shown relative to its binding to the native <i>ARS305</i> locus. The strain with no ARS in the <i>VPS13</i> locus and the native origin <i>ARS522</i> are shown as controls (<i>VPS13</i> and <i>ARS522</i>, respectively).</p