TAILIEUCHUNG - Báo cáo khoa học: 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). | ỊFEBS Journal Biochemical characterization of fragmented human MCM2 Yuki Komamura-Kohno1 Rikou Tanaka1 Akira Omori1 Toshiyuki Kohno1 and Yukio Ishimi1 2 1 Mitsubishi Kagaku Institute of Life Sciences MITILS Machida Tokyo Japan 2 Ibaraki University Mito Ibaraki Japan Keywords DNA helicase DNA replication MCM2 ssDNA binding structural domain Correspondence Y. Ishimi and T. Kohno Ibaraki University Mito Ibaraki Japan Fax 81 29 228 8439 Tel 81 29 228 8439 E-mail ishimi@ and tkohno@ Received 20 October 2007 revised 6 December 2007 accepted 12 December 2007 doi 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-termi-nal 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 .

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