Biochemical characterization of fragmented human MCM2.
The molecular dissection of human MCM2, a constituent of MCM2-7 licensing factor complex, was performed to identify the region responsible for its biochemical activities. Partial digestion with trypsin dissected the MCM2 protein into a central region (148-676) containing ATPase motifs and a C-terminal region (677-895). These two fragments, along with three other fragments (148-441, 442-676 and 442-895), were produced using the wheat germ cell-free system and were examined for their ability to inhibit MCM4/6/7 helicase activity. Two fragments (442-895 and 677-895) containing the C-terminus were partly inhibitory to the activity. Further dissection revealed that one fragment (713-895) has strong inhibitory activity. The inhibitory activity of the smaller fragments derived from the C-terminal region correlated with their ability to inhibit SV40 T antigen helicase activity and also with their ability to bind to ssDNA, which has been shown by gel mobility shift analysis. These results strongly suggest that the MCM2 fragments derived from the C-terminal region inhibit DNA helicase activity through their ability to bind to ssDNA. In contrast, two fragments (148-441 and 442-676) from the central region were mainly responsible for the interaction between MCM2 and MCM4, and this was revealed by a pulldown analysis using MCM4 protein beads. Finally, only complete MCM2, not the smaller fragments, could disassemble the MCM4/6/7 hexamer into the MCM2/4/6/7 tetramer.
Komamura-Kohno
Yuki
Y
Mitsubishi Kagaku Institute of Life Sciences (MITILS), Machida, Tokyo, Japan.
Tanaka
Rikou
R
Omori
Akira
A
Kohno
Toshiyuki
T
Ishimi
Yukio
Y
D020816:Amino Acid Motifs; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D064111:Minichromosome Maintenance Complex Component 2; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D014357:Trypsin / Q000378:metabolism
D018797:Cell Cycle Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D014357:Trypsin; D004265:DNA Helicases; C084859:MCM2 protein, human; C099243:MCM4 protein, human; C103258:MCM6 protein, human; C101624:MCM7 protein, human; D064111:Minichromosome Maintenance Complex Component 2; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7