Mcm4/6/7 complex primarily interacts with the single-stranded DNA segment
Mcm4/6/7 binds to ‘bubble’ DNA substrates and unwinds the duplex segments when the central single-stranded segment contains thymine stretches (13). In order to examine the mode of binding of Mcm4/6/7 on bubble DNAs, we conducted nuclease protection assays using labeled bubble substrate DNAs and Mcm4/6/7 in the presence of ATP-γ-S which permits binding to substrate DNA but does not mobilize the helicase. At an optimum concentration of DNase I, strong cleavages were detected on the 21 bp duplex regions at both ends of the substrates, whereas weaker cleavages were detected on the central single-stranded region, consistent with preference of DNase I to double-stranded DNA. With the Bub66/T-rich substrate, strong protection was detected on the entire single-stranded regions of both top and bottom strands with increasing concentration of Mcm4/6/7, consistent with high affinity of Mcm4/6/7 to T-rich bubble sequences (Figure 1A). Similar, but less significant protection was detected on the single-stranded DNA segment with P1 nucleases (Figure 1A), which is specific to single-stranded DNA. The results indicate that the Mcm complex is loaded onto bubble DNA through binding to both strands of the single-stranded region. Careful examination of Mcm4/6/7 footprints on a bubble indicated strong protection on the 5′-half of the top strand and moderate protection on the remaining 3′-half, as well as on several base-pair duplex segment adjacent to the branch point (Figure 1A and data not shown). A similar pattern of protection was observed also on the bottom strand. This suggests that Mcm4/6/7 may bind to a replication bubble substrate with a 2-fold symmetry as a double hexamer (Figure 1A, see the lower scheme), in a manner similar to SV40 T-antigen (21).
Nuclease protection assays were conducted also on a T-tailed Y-fork structure (Figure 1B). Not only the single-stranded tail region but also the 7 bp duplex DNA adjacent to the duplex-to-single-strand junction were protected by Mcm binding from DNaseI digestion. These results indicate that Mcm4/6/7 protein primarily interacts with single-stranded DNA region and that the interaction also spans the duplex segments near the branchpoint of the replication fork.