THESIS
2004
xiii, 100 leaves : ill. (some col.) ; 30 cm
Abstract
DNA replication is one of the most fundamental cellular processes. To ensure proper genome duplication and inheritance, eukaryotic cells exert strict control over DNA replication by regulating a series of replication-initiation proteins. Homologues of most of the replication-initiation proteins have been identified in organisms from yeast to humans. Our laboratory has recently identified Noc3p (nucleolar complex-associated protein) as a novel initiation protein and shown that it plays an integral role in the initiation of DNA replication in budding yeast. However, it has not been determined whether there is a human Noc3p homolog and whether it is required for DNA replication in human cells....[
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DNA replication is one of the most fundamental cellular processes. To ensure proper genome duplication and inheritance, eukaryotic cells exert strict control over DNA replication by regulating a series of replication-initiation proteins. Homologues of most of the replication-initiation proteins have been identified in organisms from yeast to humans. Our laboratory has recently identified Noc3p (nucleolar complex-associated protein) as a novel initiation protein and shown that it plays an integral role in the initiation of DNA replication in budding yeast. However, it has not been determined whether there is a human Noc3p homolog and whether it is required for DNA replication in human cells.
Based mainly on sequence homology, we have identified a human protein sequence called AD24 in the data base that shares significant homology with the yeast Noc3p (yNoc3p). No other human protein was found to be significantly similar to yNoc3p. Therefore, AD24 is likely to be the human Noc3p homolog.
In this project, co-immunoprecipitation experiments clearly demonstrate that AD24 and other human replication initiation proteins, such as ORC and MCM proteins have physical interactions.
In order to define the functions and mechanisms of AD24 in DNA replication in human cells, we did cell culture experiments by inhibiting the expression of the gene using the siRNA (small interfering RNA) strategy. The expression of the endogenous AD24 was inhibited with siRNA transfected into a human cell line (HeLa) to test the gene silencing effects. We found that the siRNAs efficiently and specifically inhibited the target gene expression in the HeLa cell line. The siRNAs not only significantly inhibited DNA replication of the cells as detected by using the BrdUrd incorporation assay, but also inhibited the proliferation of the cells as observed using the WST-1 assay. Furthermore, siRNAs targeted to AD24 induced apoptosis in human cancer cells, which was detected using TUNEL and Annexin-V staining assays.
To conclude, our studies show that AD24 is required for DNA replication in human cells. Inhibiting the expression of AD24 effectively abates DNA replication, thus stopping cancer cell growth. These studies help to advance our understanding of the human cell cycle and may also provide new tools for fighting cancer.
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