PMC:1140370 / 31009-34053 JSONTXT

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The archaeal Mcm can unwind A-fork[3′] but eukaryotic Mcm4/6/7 cannot, since the former binds to double-stranded DNA but the latter does not (5,27). While yeast Mcm helicases can translocate on duplex DNA, such activity was not observed with mammalian Mcm4/6/7 (data not shown).\nOccurrence of AT-rich sequences, with asymmetric distribution of adenine and thymine, near the replication origins, lead us to propose that Mcm may play a role in selection of initiation sites of mammalian DNA replication, and prompted us to examine the ability of sequences from human replication origins to activate Mcm4/6/7 helicase. Both lamin-B2 and c-myc origins served as efficient activator for Mcm helicase in vitro. Consistent with it, site-specific loading of Mcm in the DNA replication initiation zone of the c-myc was recently reported (33).\nWe have examined in detail the effect of sequence context of the single-stranded DNA on the helicase activity of Mcm. The results indicate that thymine content of 50% is sufficient for the maximum helicase activity. The efficiency of displacement decreased as the thymine content of the 3′-tail dropped to 33% (Figure 6). The stretches of thymine residues may not be necessarily required, since repeats of TC dinucleotides served as a potent activator for Mcm4/6/7. We also noticed that the presence of a secondary structure within the single-stranded DNA is inhibitory for helicase action. Nuclease footprinting assays indicated that binding was interfered by the secondary structure (data not shown). Thus, we have concluded that Mcm4/6/7 helicase is most efficiently activated by non-structured single-stranded DNA with thymine content of 50% or more, although significant stimulation is observed also by DNA with less thymine content (Figure 7), suggesting that the sequence specificity for Mcm helicase activation is rather relaxed and that the extent of the activation may depend on the sequence context. This would be reasonable given the flexibility and differential regulation of site selection for initiation of DNA replication during development or in various cell types, as well as the variability in initiation potential of each replication origin on the genome even within the single cell 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