Isolation and characterization of various complexes of the minichromosome maintenance proteins of Schizosaccharomyces pombe.
Minichromosome maintenance (Mcm) proteins 2-7 are highly conserved in eukaryotes and play an essential role in DNA replication. Here, we describe the reconstitution of the various complexes of the Mcm proteins of Schizosaccharomyces pombe using the baculovirus expression system. The simultaneous expression of all six of the Mcm proteins, as well as different combinations of these proteins, yielded several stable complexes that included the heterohexamer of Mcm2/3/4/5/6/7, the Mcm2/4/6/7 heterotetramer, the dimer of the Mcm4/6/7 heterotrimer, and the Mcm3/5 heterodimer. The purification and characterization of the biochemical properties of these complexes showed that only the dimeric complex of the Mcm4/6/7 heterotrimer possessed single stranded DNA-dependent ATPase, ATP-dependent single stranded DNA binding, and 3' to 5' DNA helicase activities. Consistent with these results, the interaction of either Mcm2 or Mcm3/5 with the Mcm4/6/7 complex resulted in the disassembly of the dimeric complex of Mcm4/6/7 and the loss of DNA helicase activity. These results suggest that the Mcm4/6/7 complex is a catalytic core of the Mcm complex and that Mcm2 and Mcm3/5 may be involved in the regulation of the activity of this complex.
Lee
J K
JK
Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Hurwitz
J
J
D001483:Base Sequence; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000378:metabolism; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D004265:DNA Helicases