Published online 25 May 2005
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DNA binding and helicase actions of mouse MCM4/6/7 helicase
Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science 18-22, Honkomagome 3-chome, Bunkyo-ku, Tokyo 113-8613, Japan
*To whom correspondence should be addressed. Tel: +81 3 5685 2264; Fax: +81 3 5685 2932; Email: hmasai{at}rinshoken.or.jp
Received March 11, 2005. Revised May 2, 2005. Accepted May 2, 2005.
Helicases play central roles in initiation and elongation of DNA replication. We previously reported that helicase and ATPase activities of the mammalian Mcm4/6/7 complex are activated specifically by thymine-rich single-stranded DNA. Here, we examined its substrate preference and helicase actions using various synthetic DNAs. On a bubble substrate, Mcm4/6/7 makes symmetric dual contacts with the 5'-proximal 25 nt single-stranded segments adjacent to the branch points, presumably generating double hexamers. Loss of thymine residues from one single-strand results in significant decrease of unwinding efficacy, suggesting that concurrent bidirectional unwinding by a single double hexameric Mcm4/6/7 may play a role in efficient unwinding of the bubble. Mcm4/6/7 binds and unwinds various fork and extension structures carrying a single-stranded 3'-tail DNA. The extent of helicase activation depends on the sequence context of the 3'-tail, and the maximum level is achieved by DNA with 50% or more thymine content. Strand displacement by Mcm4/6/7 is inhibited, as the GC content of the duplex region increases. Replacement of cytosine–guanine pairs with cytosine–inosine pairs in the duplex restored unwinding, suggesting that mammalian Mcm4/6/7 helicase has difficulties in unwinding stably base-paired duplex. Taken together, these findings reveal important features on activation and substrate preference of the eukaryotic replicative helicase.
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