| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2005: ¥2,200,000 (Direct Cost: ¥2,200,000)
|
|---|
| Research Abstract |
MCM (minichromosome maintenance) plays central roles in initiation and elongation of DNA replication as a replicative helicase. We previously reported that the helicase activity of mouse Mcm4/6/7 complex is activated specifically by thyimine-rich single-stranded DNA. In this research, 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 nucleotide 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 to various fork and extension structures with similar affinity as long as they contain a single-stranded DNA segment. Strands are displaced, however, only when it is loaded
… More
onto a single-stranded 3'-tail, permitting its translocation from 3' to 5'. 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 (You ＆ Masai, NAR 2005).
On the other hand, the Mcm helicase is expected to interact with many factors during the loading and translocation on the template DNA. Cdt1 is one of them. We found that the Mcm complex including Mcm2-7, Mcm4/6/7 and Mcm2/4/6/7 cosediment with the Cdt1 in the glycerol gradient centrifugation. Cdt1 interacts with a single polypeptide of Mcm2 as well as with the Mcm4/6 complex. Cdt1 is an ATP binding protein and forms a big complex with Mcm in the presence of ATP. The DNA binding and helicase activities of Mcm4/6/7 were dramatically stimulated by Cdt1 protein in vitro, suggesting Cdt1 facilitates the loading of Mcm onto chromatin. These facts suggest that Cdt1 has a similar function to DnaC that is the loading factor of E.coli DnaB helicase. Less
|
