DUE sequences from the c-myc origin activate Mcm helicase
Our previous studies have shown that the sequences containing periodic (dT)n tracts derived from the human lamin B2 origin (48% thymine content) can serve as an efficient activator for the Mcm helicase on a bubble, and replacement of thymines with guanines abolished the helicase activation (13,28). In order to examine other natural replication origins with different thymine contents for the ability to activate Mcm helicase on a bubble substrate, we constructed new bubble substrates, Bub82/c-myc, containing sequences derived from the DUE (DNA unwinding element) region of the human c-myc origin which is essential for c-myc replicator activity (29,30). The unpaired segment in Bub82/c-myc/DUE contains one strand of DUE and that in Bub82/c-myc/DUE-C contains another strand. This DUE is believed to coincide with the initially unwound region of the c-myc origin (29), and its deletion substantially reduced the replicator activity (30). Mcm4/6/7 unwound Bub82/lamin B2 (thymine 48%), Bub82/c-myc/DUE (37%) and Bub82/c-myc/DUE-C (39%) with similar efficiency (Figure 7A), indicating that the sequences from the c-myc origin also can serve as efficient activators of the Mcm helicase. This result also indicates that sequences with 37% thymine content can activate Mcm4/6/7 to displace 24 nt duplex on both sides in a bubble substrate, suggesting that the not only thymine content but also the sequence context may affect the Mcm helicase activation.
In gel shift assays, MCM4/6/7 bound to all the three bubbles, although the affinity to Bub82/c-myc/DUE and to Bub82/c-myc/DUE-C is slightly lower than that to Bub-82/lamin B2 (Figure 7B). Therefore, helicase and DNA binding activities do not necessarily correlate with each other. This was also indicated by our previous results that guanine and cytosine stretches can bind to Mcm but cannot activate the helicase (13).