1. Chromatin fractionation analysis of licensing factors in mammalian cells.
- Author
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Nishitani H, Morino M, Murakami Y, Maeda T, and Shiomi Y
- Subjects
- Cell Culture Techniques methods, Cell Cycle, Cell Cycle Proteins metabolism, Chromatin metabolism, HeLa Cells, Humans, Minichromosome Maintenance Proteins isolation & purification, Minichromosome Maintenance Proteins metabolism, Protein Binding, Cell Cycle Proteins isolation & purification, Chemical Fractionation methods, Chromatin isolation & purification
- Abstract
ORC, Cdc6, Cdt1, and MCM2-7 are replication-licensing factors, which play a central role in the once-per-cell cycle control of DNA replication. ORC, Cdc6, and Cdt1 collaborate to load MCM2-7 onto replication origins in order to license them for replication. MCM2-7 is a DNA helicase directly involved in DNA replication and dissociates from DNA as S phase progresses and each replicon is replicated. In the cell cycle, the loading of MCM2-7 is restricted during the end of mitosis and the G1 phase. Thus, the levels of chromatin-bound MCM2-7 and its loaders oscillate during the cell cycle. Chromatin association of these factors can be analyzed by separating a cell lysate into soluble and chromatin-enriched insoluble fractions in mammalian cells.
- Published
- 2014
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