DNA binding and helicase actions of mouse MCM4/6/7 helicase.
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.
You
Zhiying
Z
Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science 18-22, Honkomagome 3-chome, Bunkyo-ku, Tokyo 113-8613, Japan.
Masai
Hisao
H
D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D020862:GC Rich Sequence; D016259:Genes, myc; D051379:Mice; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000378:metabolism*; D011071:Poly T / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013941:Thymine / Q000032:analysis
D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011071:Poly T; D004265:DNA Helicases; C491205:Mcm6 protein, mouse; C491086:Mcm7 protein, mouse; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; D013941:Thymine