I am having difficulty answering three homework questions which relate directly to Chong et al. (2000).
The authors have determined that MtMCM is able to bind both ssDNA and dsDNA (see Figure 1). However, they don’t mention whether it is the same or a different site on the protein that is involved in binding the two forms of DNA.
Question #1: Propose an experiment that would help to distinguish between these two possibilities and what result would you expect?
Question #2: If ssDNA and dsDNA do share the same MtMCM binding site, what part of the DNA do you suppose is involved in stimulating the ATPase activity? In a helicase assay, the authors show that MtMCM can displace large ssDNA fragments (up to 500 bases) from a circular substrate (p. 1533).
Question #3: Explain why this is, or is not, a suitable assay for MtMCM processivity?
One possible method to determine whether the MtMCM complex uses two different sites to bind ssDNA and dsDNA is through the identification and blockage (i.e. by using a ligand) of a single specific binding site on the MtMCM. If the blockage of this site prevents the binding of MtMCM to both ssDNA and dsDNA, this would suggest that the same site is used for both. The research paper suggests that ssDNA and dsDNA do share the same binding site because chelation of magnesium ions prevented the protein from binding to DNA.
No ATP hydrolysis was detected in reaction mixtures containing the K341E or delta-N mutant proteins, however, both wt and K341E protein still formed complexes with the DNA. If ssDNA and dsDNA do share the same MtMCM binding site, then perhaps it’s the delta-N portion of the DNA that is involved in stimulating ATPase activity.
I found it hard to start this question. This is what I have so far: This is a suitable assay for MtMCM processivity because a population of substrates possessing displaceable strands of increasing length was used. This allowed the researchers to compare the displacement of the different strands and quantify the processivity of the MCM complex.
Let me know if I should provide more information. Any help or guidance would be greaetly appreciated!