PMC:1140370 / 20617-22640 JSONTXT

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Increase of thymine content on the 3′-tail did not further increase the extent of unwinding (37-TTTA13, 37-TTTTA10 and 37-T50; Figure 6A), suggesting that 17 repeats of TT dinucleotides (67% thymine) may be sufficient for activation of Mcm helicase. It should be noted that the TA25 tail would self-anneal and this would prevent the loading of Mcm, since the 3′-tail containing TTAA13 or TTTAAA8 did not enhance the unwinding either (data not shown). Therefore, we next examined the effects of repeating sequences of thymine and cytosine (Figure 6B). Partial heteroduplex substrates containing the 3′-tail of TC25, TTCC13, TTTCCC8, TTTTCCCC6, TTCC17 were displaced by the Mcm4/6/7 complex to the extent similar to that achieved by TTA17, suggesting that 50% thymine content is sufficient for full activation of Mcm helicase. TTCC13 and TTCC17 were displaced to a similar extent, consistent with the earlier result that a 40 nt tail is sufficient for activation (13). However, the efficiency of the displacement significantly decreased as the thymine content of the 3′-tail dropped to 33%, as in TAA17 or TCC17 (Figure 6B and C). Thus, the minimal thymine content required for activation of the Mcm helicase may be somewhere between 33 and 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