BMCB625:DNA Replication (New components)

(Homework) Questions

Jon

In regards to figure 4A, why does the helicase not knock off the 3' label?

In contrast to the figure 5B the only logical answer is that the helicases translocates the DNA in the 3' to 5' direction and must assemble on the single stranded DNA (not the double stranded as shown in the misleading figure 5B).

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Chris

Numerous studies have suggested that the Mcm2-7 complex does not display helicase activity in vitro, however individual subcomplexes (namely Mcm-4, -6, & -7) do display helicase activity.

Also, Kearsy and Labib (1998) propesed that MCM2-7 proteins bind to chromatin in a cell-cycle dependent manner (being tightly bound in late mitosis and G1; being removed in S- and G2-phase) (Maiorano et al, Kearsy and Labib).

Why might these features be beneficial during replication of chromosomes in eukaryotes? Hint: what is the proposed function of this entire complex in the Moyer paper.

--Chris 12:47, 17 April 2007 (EDT)

ANSWER: This regulation would allow for a potential mechanism in controlling the replication of origin firing. It is absolutely critical to restrict replication of the chromosome to only one round per cell cycle (Domenico, Lutzmann, Mechali, Curr. Opin. Cell Biol. (2006)v18:130-136)

--Chris 16:17, 22 April 2007 (EDT)

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Mahta

Q1. What do you think is the best way to determine the role of post-translational modifications in the formation of the CMG complex / activation of the helicase?

A1. One possible way: purify the components separately post-S phase (G2) and also in the complex (as in the paper). Components purified in G2 should not contain the PTMs that cause association. Analyze via 2D. PTMs such as phosphorylation are easy to detect by this method - it can also be combined with tandem MS. To further test phosphorylation of components they can be treated with phosphatases...

Q2. MCM4/6/7 have helicase activity together in vitro. MCM2/3/5 may be playing more of inhibitory roles. Therefore, aren't 2 MCMs enough? (ie, you could have a trimer of MCM4 for helicase activity and a trimer of MCM2 for inhibition). What does the system gain by having so many MCMs?

A2. Eukaryotic chromosomes contain many replication origins. Do they all take on the same structures upon replication initiation? Perhaps one MCM over another can recognize a specific structure (they do appear along the lengths of chromosomes)and serve as a scaffold for recruiting other MCMs and the rest of the CMG complex.

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Jeremy

1. If the purification strategy in figure 1A hadn't been so stringent, what other kinds of factors might of been isolated with cdc45?

The stringent purification used in Moyer et al not suprisingly isolated a core helicase complex. A less stringent approach may have identified new components of the replication or pre-replication machinery like cdt-1. Mass spectrometry based approaches to identify new protein partners or complexes is a compromise between isolating tightly bound proteins or loose or transient partners, the latter can often produce false positives since this requires gentle conditions.

2. What does figure 5 prove?

Figure 5 demonstrates that cdc45 is necessary for S-phase progression. The others suggest this shows that cdc45 is a necessary component of the replication machinery in vivo, but cdc45 may also trigger the S/M cell cycle check point. Further experiments in vivo are necessary to prove that cdc45 is a component of the replication machinery. Co-localization, for example, of cdc45 using replication specific antibodies towards polII on a replicating chromosome may provide further evidence.

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Larry

According to current understanding, what is the most plausible mechanism for CMG component interaction and how could this be tested?

Answer: Phosphorylation by cdks. Could be tested by site-directed mutagenesis of CMG components to eliminate phosphorylation sites and see if interaction remains.

Larry's Question Page

--LarryGray 11:18, 19 April 2007 (EDT)