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 × 10(7) 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.

Chromatin-dependent and -independent regulation of DNA replication origin activation in budding yeastEfficient activation of early DNA replication origins in S phase depends on the binding of Forkhead transcription factors and is independent of the chromatin environment.

DNA replication origins are licensed in early G1 phase of the cell cycle where the origin recognition complex (ORC) recruits the minichromosome maintenance (MCM) helicase to origins. These pre-replicative complexes (pre-RCs) remain inactive until replication is initiated in the S phase. However, transcriptional activity in the regions of origins can eliminate their functionality by displacing the components of pre-RC from DNA. We analyzed genome-wide data of mRNA and cryptic unstable transcripts in the context of locations of replication origins in yeast genome and found that at least one-third of the origins are transcribed and therefore might be inactivated by transcription. When investigating the fate of transcriptionally inactivated origins, we found that replication origins were repetitively licensed in G1 to reestablish their functionality after transcription. We propose that reloading of pre-RC components in G1 might be utilized for the maintenance of sufficient number of competent origins for efficient initiation of DNA replication in S phase.

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.In order to produce mRNA, RNA polymerase II (RNAPII) has to contend with chromatin in regulatory and coding regions of genes. Different modifications of histone proteins, rearrangements of nucleosome positioning, and packaging of nucleosomes into higher-order chromatin structures are used to facilitate or repress the accessibility of cellular factors to DNA. Modifications of histone proteins by acetylation and methylation are the most common ways to initiate changes in chromatin structure. In addition to several transcription-coupled modifications of chromatin in active gene loci, nucleosomes can be removed from entire transcribed regions and replaced with new histones after shutdown of transcription (14,18,21,42,49,54). A detailed study of nucleosome dynamics in budding yeast has revealed a remarkable replication-independent exchange of histones throughout the genome. While turnover of promoter nucleosomes is detectable regardless of transcriptional activity of the locus, exchange of histones in the coding regions is in good correlation with gene expression level, indicating that turnover of nucleosomes is a rather common feature of ongoing transcription, especially in highly transcribed loci (7).Gene expression in eukaryotic cells is also controlled by the formation of repressive heterochromatic domains in loci of regulated genes. There are three main regions in the genome of Saccharomyces cerevisiae that are subject to silencing by heterochromatin: telomeres, ribosomal DNA (rDNA) locus, and silent mating type loci (HML and HMR). Heterochromatin at telomeres and HM loci is formed by the complex of silent information regulator (SIR) proteins Sir2, Sir3, and Sir4, which are also required f...

Protocol Summary 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.