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 c ontain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. Our group has reported that a DNA helicase ac tivity is associated with a 600 kDa human Mcm4, 6 and 7 complex. The struct ure of the Mcm4,6,7 complex was visualized by electron microscopy after neg ative staining with uranyl acetate. The complex contained toroidal forms wi th a central channel and also contained structures with a slit. Gel-shift a nalysis indicated that the level of affinity of the Mcm4,6,7 complex for si ngle-stranded DNA was comparable to that of SV40 T antigen, although the Mc m4,6,7 complex required longer single-stranded DNA for the binding than did SV40 T antigen. The nucleoprotein complexes of Mcm4,6,7 and single-strande d DNA were visualized as beads in a queue or beads on string-like structure s. The formation of these nucleoprotein complexes was erased by Mcm2 that i s a potential inhibitor of the Mcm4,6,7 helicase. We also found that the DN A helicase activity of Mcm4,6,7 complex was inhibited by the binding of Mcm 3,5 complex. These results support the notion that the Mcm4,6,7 complex fun ctions as a DNA helicase and the formation of 600 kDa complex is essential for the activity. (C) 2000 Academic Press.