|Budget Amount *help
¥6,500,000 (Direct Cost : ¥6,500,000)
Fiscal Year 1998 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1997 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 1996 : ¥3,200,000 (Direct Cost : ¥3,200,000)
1. Studies on chromatin proteins interacting with MCM proteins.
(1) We have identified five proteins physically associated with XMcm3 as XMcm2, 4, 5, 6, 7 proteins by analyzing the immunoprecipitates of egg extract with anti-XMcm3 antibody. (2) XMCM complex could be dissociated into subcomplexes, consisting of XMcm3-5, and XMcm2-4-6-7. (3) we have identified histon H3 physically interacted with XMcm3 by far-western, and pull down experiments. (4) We have identified Xenopus homolog of budding yeast Cdc45, which interacts with MOM proteins, and found that XCdc45 interacted with DNA polymerase alpha in the egg extract.
2. Identification of proteins involved in the initiation of DNA replication, and clarification of its possible role in the replication.
In Xenopus egg extract, XMCM protein complex consisting of 6 members of XMCM proteins was required for the initiation of replication, and subcomplex consisting of XMcrn3-5 and XMcm2-4-6-7 could not substitute for it.
3. Identification of chromatin structures competent to the initiation of DNA replication.
(1) all six XMcm proteins associated with chromatin before the formation of nuclei, and they were released from it upon the progression of DNA replication. (2) XMcm3 proteins associated with chromatin could not be released when the chromatin was condensed upon the addition of M-phase egg extract. (3) XCdc45 became associated with chromatin after the formation of nuclei, and its binding to chromatin is essentially required for the loading of DNA polymerase alpha onto chromatin.