S phase is a phase of the cell cycle, which consists of G1 phase, S phase, G2 phase, and finally mitosis where the cell divides to make two daughter cells. During S (which stands for synthesis) phase the entire DNA content of the cell is duplicated. The copied DNA will then be partitioned into two daughter cells during mitosis. Cells enter this phase after signals during G1 phase indicate that the internal and external environments are ideal for DNA replication. Major proteins involved in this transition are retinoblastoma (Rb), cdk2, and cyclin E.

The timing and organization of S phase are tightly controlled. The phase is just long enough for the replication machinery to create only one copy of all DNA. Additionally, certain blocks are in place to prevent additional copies from accidentally being made. Certain regions of DNA also have a certain order for replication. For example, housekeeping genes, which are active in all cells, are always replicated early in the phase, while highly condensed regions of chromosomes are replicated later. Overall, the time a cell takes to get through S phase varies greatly depending on the species and type of cell. Most kinds of dividing human cells generally take about 9 hours.

At any given time, a certain population of cells growing in tissue culture will be in various stages of the cell cycle. There are two common methods for determining the percentage of cells that is in S phase. First, the cells can be treated for a short period of time with the radiolabeled nucleotide 3H-thymidine. Cells that are in S phase will incorporate this nucleotide into their DNA, making them radioactive. The amount of radioactivity can then be determined to find the percentage of cells in S phase. Likewise, the cells can be treated with BrdU, a nonradioactive thymidine analog that it also incorporated into DNA and can be detected by an antibody. Second, the cells can be treated with a DNA dye, such as Hoechst, and analyzed by flow cytometry. (See Hoechst dye for more detail on how this works.)

Cells in tissue culture can be arrested specifically in S phase by treating them with certain chemicals that inhibit DNA replication. Some examples include aphdicolin, a DNA polymerase inhibitor, and hydroxyurea, an inhibitor of deoxyribonucleotide synthesis. This arrest can be reversed so the cells can continue through the cycle simply by removing the chemicals.

Albert's Molecular Biology of the Cell, 3rd edition.
Current Protocols in Cell Biology, Volume 1, Section 8

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