Mapping Critical DNA Sequence Elements Required for Amplification of erbB2 in Breast Cancer
Abstract
Recent studies show that 80-90% of breast carcinomas are associated with gene amplification. Each amplicon most likely has an origin of replication. In Drosophila, DmORC (Origin Recognition Complex) binds to specific DNA sequences and this binding is essential for chorion gene amplification. To study the implications of replication initiator proteins to gene amplification, we characterized two proteins required for DNA replication (ORC2 and Mcml0) . We created hypomorphic mutation in the ORC2 gene of a cancer cell line through homologous recombination. We found that these cells failed to support the replication of an extrachromosomal plasmid bearing the oriP replicator of Epstein Barr virus (EBV) . Geminin, an inhibitor of replication initiation complex, inhibited replication of this episome. The result identifies a novel means by which to cure cancer with gene amplification where the amplicons are carried as episomes. We also report that Xenopus Mcml0 (XMcml0) is not required for origin binding of XMcm2-7. Instead, the chromatin binding of XMcml0 at the onset of DNA replication requires chromatin-bound XMcm2-7, and it is independent of Cdk2 and Cdc7. In the absence of XMcml0, XCdc45 binding, XRPA binding, and initiation-dependent plasmid supercoiling are blocked. Therefore, XMcml0 performs its function after pre-RC assembly and before origin unwinding.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 01, 2002
- Accession Number
- ADA406150
Entities
People
- Anindya Dutta
- Yuichi Machida
Organizations
- Brigham and Women's Hospital