Electron microscopic observation and single-stranded DNA binding activity of the Mcm4,6,7 complex.
Mcm2-7 proteins that play an essential role in eukaryotic DNA replication contain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. Our group has reported that a DNA helicase activity is associated with a 600 kDa human Mcm4, 6 and 7 complex. The structure of the Mcm4,6,7 complex was visualized by electron microscopy after negative staining with uranyl acetate. The complex contained toroidal forms with a central channel and also contained structures with a slit. Gel-shift analysis indicated that the level of affinity of the Mcm4,6,7 complex for single-stranded DNA was comparable to that of SV40 T antigen, although the Mcm4,6,7 complex required longer single-stranded DNA for the binding than did SV40 T antigen. The nucleoprotein complexes of Mcm4,6,7 and single-stranded DNA were visualized as beads in a queue or beads on string-like structures. The formation of these nucleoprotein complexes was erased by Mcm2 that is a potential inhibitor of the Mcm4,6,7 helicase. We also found that the DNA helicase activity of Mcm4,6,7 complex was inhibited by the binding of Mcm3,5 complex. These results support the notion that the Mcm4,6,7 complex functions as a DNA helicase and the formation of 600 kDa complex is essential for the activity.
Sato
M
M
Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Tokyo, Machida, 194-8511, Japan.
Gotow
T
T
You
Z
Z
Komamura-Kohno
Y
Y
Uchiyama
Y
Y
Yabuta
N
N
Nojima
H
H
Ishimi
Y
Y
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D002868:Chromosomal Proteins, Non-Histone; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008854:Microscopy, Electron; D064111:Minichromosome Maintenance Complex Component 2; D064126:Minichromosome Maintenance Complex Component 3; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D009942:Organometallic Compounds; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D016626:Shadowing Technique, Histology; D013379:Substrate Specificity
D000952:Antigens, Polyomavirus Transforming; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C066470:MCM3 protein, human; C513269:Mcm3 protein, mouse; D009687:Nuclear Proteins; D009942:Organometallic Compounds; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C085711:mcm5 protein, S pombe; C005460:uranyl acetate; D004265:DNA Helicases; C092802:MCM4 protein, S cerevisiae; C491207:MCM6 protein, S cerevisiae; C103258:MCM6 protein, human; C491088:MCM7 protein, S cerevisiae; C101624:MCM7 protein, human; C491205:Mcm6 protein, mouse; C491086:Mcm7 protein, mouse; D064111:Minichromosome Maintenance Complex Component 2; D064126:Minichromosome Maintenance Complex Component 3; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7