Histone variants: The unsung guardians of the genome.

Histones H2A, H2B, H3, H4 and H1 are highly conserved, positively charged proteins which form a disc-shaped protein core around which genomic DNA is wrapped to form a nucleosome. Immediately following DNA synthesis, replication-dependent canonical histones help package the DNA into nucleosomes to form compact chromatin fibers that can fit within the confines of the cell nucleus. Histone variants, which vary from the canonical histones in their primary amino acid sequence and expression patterns, replace their canonical counterparts throughout the cell cycle in important biological processes such as transcription, replication, DNA repair and heterochromatin formation. DNA damage is a continual threat to genomic stability and cell survival. Unrepaired DNA lesions are either lethal or can promote mutations if the damaged cells escape programmed cell death due to apoptosis. In order to repair DNA damage, cells use multiple DNA repair pathways, all of which require the recruitment of a multiple DNA damage signaling and repair factors. In order for these repair factors to be recruited efficiently and function properly at sites of DNA damage, the local chromatin environment surrounding the DNA lesion is often altered. Cells are able to regulate chromatin structure in the vicinity of DNA lesions through the addition of posttranslational modifications on histones and DNA, as well as through histone variant incorporation or removal. Recruitment or removal of histone variants at sites of DNA damage can alter the local chromatin structure by destabilizing it and making it more accessible to repair factors. Alternatively, some histone variants and their modifications may also provide specific binding sites for the recruitment of various DNA repair factors, thereby influencing repair pathway choice or repair efficiency, or both. This review seeks to provide an overview of our current understanding of the roles played by histone variants in DNA repair, especially in mammalian cells.

Preparation of Cell Extracts by Cryogrinding in an Automated Freezer Mill.

The ease of genetic manipulation and the strong evolutionary conservation of eukaryotic cellular machinery in the budding yeast Saccharomyces cerevisiae has made it a pre-eminent genetic model organism. However, since efficient protein isolation depends upon optimal disruption of cells, the use of yeast for biochemical analysis of cellular proteins is hampered by its cell wall which is expensive to digest enzymatically (using lyticase or zymolyase), and difficult to disrupt mechanically (using a traditional bead beater, a French press or a coffee grinder) without causing heating of samples, which in turn causes protein denaturation and degradation. Although manual grinding of yeast cells under liquid nitrogen (LN2) using a mortar and pestle avoids overheating of samples, it is labor intensive and subject to variability in cell lysis between operators. For many years, we have been successfully preparing high quality yeast extracts using cryogrinding of cells in an automated freezer mill. The temperature of -196 °C achieved with the use of LN2 protects the biological material from degradation by proteases and nucleases, allowing the retrieval of intact proteins, nucleic acids and other macromolecules. Here we describe this technique in detail for budding yeast cells which involves first freezing a suspension of cells in a lysis buffer through its dropwise addition into LN2 to generate frozen droplets of cells known as "popcorn". This popcorn is then pulverized under LN2 in a freezer mill to generate a frozen "powdered" extract which is thawed slowly and clarified by centrifugation to remove insoluble debris. The resulting extracts are ready for downstream applications, such as protein or nucleic acid purification, proteomic analyses, or co-immunoprecipitation studies. This technique is widely applicable for cell extract preparation from a variety of microorganisms, plant and animal tissues, marine specimens including corals, as well as isolating DNA/RNA from forensic and permafrost fossil specimens.