Replicative DNA polymerases require a ssDNA template, yet polymerases themselves lack dsDNA unwinding activity (Kawasaki and Sugino, 2001). Enter the helicase, a motor protein that uses NTP-dependent conformational changes to unwind duplex DNA by separating two annealed nucleic acid strands by breaking the hydrogen bonds between them. The heterohexameric complex known as the Mcm2-7 (minichromosome maintenance) complex has been presumed, for the last 15 years, to serve as this helicase in mammalian cells (Bochman and Schwacha, 2009), with recent recombinant in vitro experiments demonstrating that Mcm2-7 unwinds DNA, either alone or within the context of a larger complex (Bochman and Schwacha, 2008). To date, however, only data from studies done in non-physicolical settings exist concerning the mammalian Mcm2-7 proteins; they have never before been studied in a live cell mammalian setting, and so the true nature of Mcm2-7's interaction with chromatin has not been clear. Is it dyanamic like other replication associated proteins (Leonhardt et al., 2000; McNairn et al., 2005; Xouri et al., 2007), or stable like core histones (Kanda et al., 1998; Kimura and Cook, 2001)? Afterall, both classes of these proteins interact with DNA. Given that Mcm binding to chromatin marks the final step in the formation of the pre-replication complex (pre-RC) in the eukaryotic G1 phase of the cell cycle (a process referred to as "replication licensing" (Prasanth et al., 2004)), and that DNA replication is stringently controlled to ensure replication takes place just once during the cell cycle, some dyanamic shift should take place around the time of late mitosis/early G1, when Mcms load onto chromatin, through S phase, when Mcms clear from chromatin. To test this, we employed Fluoresence Recovery After Photobleaching (FRAP) as a tool to detect chromatin interactions in living cells, and found that the Mcms do show significant biophysical changes in their interactions with chromatin throughout the cell cycle, and that they do not exchange on and off chromatin during S phase.
