Questions for Packet #1
If you would like to view the photos in the Melixetian paper in color, please see the on-line link.
Machida, Y, and A Dutta. Minireview: Cellular checkpoint mechanisms monitoring proper initiation
of DNA replication. JBC Papers in Press. Dec. 9 2004.
Melixetian, M, A Ballabeni, L Masiero, P Gasparini, R Zamponi, J Bartek, J. Lukas, and K Helin.
Loss of Geminin induces rereplication in the presence of functional p53. J. Cell Biol. 165(4): 473482, May 24, 2004.
Please define the following with respect to their use in these papers (2 points each):
Origin of replication
Licensed for replication
Ready to fire
Nuclear exclusion
Xenopus
Orthologues
P53 (as it relates to the cell cycle)
Please type answers to the following questions
Machida and Dutta
1. (4 points) How is it thought that cells typically prevent re-replication of their DNA? What are the
major proteins involved, and how does the process work?
Melixetian et al.
2. (8 points) In Figure 1C, the authors indicate that the results show that there is 4N and greater DNA
present.
a) How do they interpret this from these figures? Make a rough drawing of the figure and indicate
where the cells are that have 4N DNA?
b)What is BrdU and why are they measuring its incorporation? What relevance does the level of
cyclin B1 have to these results?
3. (4 points) How does nocodazole synchronize cells in mitosis? Why did the authors need to have
synchronized cells?
4. (4 points) The authors claim that U2OS cells with HA-tagged alterations in the nucleotide code for
geminin, when treated with siRNA to geminin lead to a strong reduction in the number of cells
with 4N and greater DNA content.
a) What is an HA tag?
b) Where is this evidence of reduction in DNA content shown? Do you agree with these findings?
Why or why not?
5. (2 points) What is the difference between an “early firing” and a “late firing” replication origin?
Why do the experimenters want to monitor this? What do they find?
6. (6 points) What is the technique called FISH? Explain how it works. How did this technique help
the authors to determine that newly synthesized DNA “heritable silenced genes” and
“heterochromatic domains” separate poorly because of sister chromatid cohesion?
7. (5 points) How does the DNA damage checkpoint in the cell cycle work? Explain proteins
involved and the sequence of events. Why did the authors examine H2AX and RAD51?
8. (6 points) When the checkpoint at CHK1 is inhibited, what happens to the cells? What
happens when they eliminate p53 function? What do they conclude from these experiments?
9. (4 points) What are CDT1 and CDC6? What did the researchers do to determine whether they
played a role in rereplication upon loss of geminin?
10. (2 points) Do you think that a mutation in geminin could be responsible for some types of
cancer? Explain why or why not.