| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2000: ¥3,900,000 (Direct Cost: ¥3,900,000)
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| Research Abstract |
The Bloom syndrome is a genetic disorder characterized by cancer-prone symptoms and is caused by mutation of the BLM gene, encoding DNA helicase that belongs to a RecQ helicase family. The aim of this study is to understand the functions of BLM helicase in DNA replication by a biochemical investigation of its function using in vitro DNA replication system.
We performed SV40 DNA replication with the cell extracts prepared from the Bloom syndrome (BS) cells as well as normal human cells. When reactions were carried out under the conditions that allow only replication, any difference in replication products from normal and BS cell extracts was seen. On the other hand, when the reaction that allows replication-coupled chromatin assembly was performed, fragmentation of replicated chromatin was seen only in BS cell extracts. To confirm that chromatin fragmentation was due to the absence of functional BLM, we expressed and purified recombinant BLM using the baculovirus-expression system. However, because we encountered a difficulty in maintaining the BS cell lines during this research, it became impossible to continue the experiments with the BS cell extracts.
Thus, we switched our experimental system to that with Xenopus egg extracts. First, we cloned a cDNA encoding a Xenopus homolog of BLM helicase and prepared specific antibodies that recognized Xenopus BLM in egg extracts. It was found that chromatin binding of Xenopus BLM during replication was slightly increased when DNA replication was arrested by DNA polymerase inhibitor, aphidicolin, and also when DNA double-strand breaks were introduced in chromatin template. In addition, it was also shown that a significant amount of BLM still bound to chromatin even when replication initiation was inhibited by geminine. These results imply that BLM may play some roles other than those at stalled replication forks and double-strand breaks.
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