of DNA replication and its linkage to cell division in all living organisms on planet Earth Genome Duplication is a comprehensive and readable textbook for advanced undergraduates, graduate students and professional scientists. It describes the underlying principles governing genome duplication from the simplest single cell bacterium to the most complex multicellular organism. It includes concepts, mechanisms, evolution and disease. 2. Studies on the Origin Recognition Complex that determines where DNA replication begins Initiation of eukaryotic genome duplication begins when a six-subunit origin recognition complex (ORC) binds to DNA. However, the mechanism by which this occurs in vivo and the roles played by individual subunits appear to differ significantly among organisms. Previous studies identified a soluble human ORC(2-5) complex in the nucleus, an ORC(1-5) complex bound to chromatin, and an Orc6 protein that binds weakly, if at all, to other ORC subunits. Here we show that stable ORC(1-6) complexes also can be purified from human cell extracts and that Orc6 and Orc1 each contain a single nuclear localization signal that is essential for nuclear localization but not for ORC assembly. The Orc6 nuclear localization signal, which is essential for Orc6 function, is facilitated by phosphorylation at its cyclin-dependent kinase consensus site and by association with Kpna6/1, nuclear transport proteins that did not co-purify with other ORC subunits. These and other results support a model in which Orc6, Orc1, and ORC(2-5) are transported independently to the nucleus where they can either assemble into ORC(1-6) or function individually. 3. Development of a high throughput screening assay for small molecules that induce DNA re-replication in human cells Previous studies have shown DNA re-replication can be induced in cells derived from human cancers under conditions in which it is not possible for cells derived from normal tissues. Because DNA re-replication induces cell death, this strategy could be applied to the discovery of potential anticancer therapeutics. Therefore, an imaging assay amenable to high-throughput screening was developed that measures DNA replication in excess of four genomic equivalents in the nuclei of intact cells and indexes cell proliferation. This assay was validated by screening a library of 1,280 bioactive molecules on both normal and tumor-derived cells where it proved more sensitive than current methods for detecting excess DNA replication. This screen identified known inducers of excess DNA replication, such as inhibitors of microtubule dynamics, and novel compounds that induced excess DNA replication in both normal and cancer cells. In addition, two compounds were identified that induced excess DNA replication selectively in cancer cells and one that induced endocycles selectively in cancer cells. Thus, this assay provides a new approach to the discovery of compounds useful for investigating the regulation of genome duplication and for the treatment of cancer. 4. CHK1, the protein kinase that induces cell cycle exit in response to DNA damage can also prevent cell cycle exit in the absence of DNA damage. Trophoblast stem (TS) cells proliferate in the presence of fibroblast growth factor-4, but in its absence, they differentiate into polyploid trophoblast giant (TG) cells that remain viable but nonproliferative. Differentiation is coincident with expression of the CDK-specific inhibitors p21 and p57;of which p57 is essential for switching from mitotic cell cycles to endocycles. Here we show that, in the absence of induced DNA damage, checkpoint kinase-1 (CHK1), an enzyme essential for preventing mitosis in response to DNA damage, functions as a mitogen-dependent protein kinase that prevents premature differentiation of TS cells into TG cells by suppressing expression of p21 and p57, but not p27, the CDK-inhibitor that regulates mitotic cell cycles. CHK1 phosphorylates p21 and p57 proteins at specific sites, thereby targeting them for degradation by the 26S proteasome. TG cells lack CHK1, and restoring CHK1 activity in TG cells suppresses expression of p57 and restores mitosis. Thus, CHK1 is part of a 'G2 restriction point'that prevents premature cell cycle exit in cells programmed for terminal differentiation, a role that CHK2 cannot play.

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DePamphilis, Melvin L (2016) Genome Duplication: The Heartbeat of Developing Organisms. Curr Top Dev Biol 116:201-29
Vassilev, Alex; Lee, Chrissie Y; Vassilev, Boris et al. (2016) Identification of genes that are essential to restrict genome duplication to once per cell division. Oncotarget 7:34956-76
DePamphilis, M L (2016) Genome Duplication at the Beginning of Mammalian Development. Curr Top Dev Biol 120:55-102
Huang, Yi-Yuan; Kaneko, Kotaro J; Pan, Haiyan et al. (2015) Geminin Is Essential to Prevent DNA Re-Replication-Dependent Apoptosis in Pluripotent Cells, but not in Differentiated Cells. Stem Cells :
Hao, Jing; de Renty, Christelle; Li, Yongming et al. (2015) And-1 coordinates with Claspin for efficient Chk1 activation in response to replication stress. EMBO J 34:2096-110
de Renty, Christelle; DePamphilis, Melvin L; Ullah, Zakir (2014) Cytoplasmic localization of p21 protects trophoblast giant cells from DNA damage induced apoptosis. PLoS One 9:e97434
Kaneko, Kotaro J; Depamphilis, Melvin L (2013) TEAD4 establishes the energy homeostasis essential for blastocoel formation. Development 140:3680-90
Zielke, Norman; Edgar, Bruce A; DePamphilis, Melvin L (2013) Endoreplication. Cold Spring Harb Perspect Biol 5:a012948
Depamphilis, Melvin L; de Renty, Christelle M; Ullah, Zakir et al. (2012) ""The Octet"": Eight Protein Kinases that Control Mammalian DNA Replication. Front Physiol 3:368
Li, Yongming; Xiao, Haijie; de Renty, Christelle et al. (2012) The involvement of acidic nucleoplasmic DNA-binding protein (And-1) in the regulation of prereplicative complex (pre-RC) assembly in human cells. J Biol Chem 287:42469-79

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