DNA replication occurs during S phase of the cell cycle. Initiation of DNA replication occurs at origins. There are a number of origins and initiation of replication at these origins is said to be spatially and temporally regulated. This has been previously shown in an experiment involving bromodeoxyuridnine (BrdU) a thymidine analogue. When DNA is replicated in the presence of BrdU it is incorporated into the DNA double helix instead of thymidine. Cells were grown in the presence of BrdU and so incorporated BrdU in their DNA. Thymidine was added at different stages of S phase to the cells. The section of the chromosome that was at that time being replicated incorporated thymidine into the double helix instead. When examined under the microscope, the sites that were replicating at the time of thymidine addition light up. These were different for early, mid and late S phase REF KELLIE/ MOLCELL BOOK. If certain proteins in this case Rif1 were localised at these origins at the time when replication is occurring perhaps then they could be involved in determining where and when replication occurs, this is why deciphering the localisation is key in both papers.
The localisation was established in two different ways. Rif1 was found to be bound to the insoluble nuclear structures in
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In any Western blot it is essential to use loading controls. They can confirm the same amount of protein was loaded in each well and also can be used as a comparison for decreased or increased protein expression of the protein being studied. For example in the Yamazaki paper Fig4a shows Rif1 expression to be similar to that of LaminB1 a nuclear protein, and Histone 3 protein also in the nucleus which is involved in creating the nucleosome structure. Tubulin is a protein located in the cytoplasm its expression does not overlap with Rif1. This allowed the authors to suggest that Rif1 was located in the nuclear-insoluble