Highlights d ORC self-interacts and dimerizes at the M-to-G 1 transition and de-dimerizes in S phase d ORC dimers provide symmetric platforms to load the symmetric pre-RCs at origins d Upon ORC de-dimerization, each single hexamer binds one of the two nascent origins d ORC dimerization is essential for and regulates pre-RC formation and DNA replication

Noc3p (Nucleolar Complex-associated protein) is an essential protein in budding yeast DNA replication licensing. Noc3p mediates the loading of Cdc6p and MCM proteins onto replication origins during the M-to-G 1 transition by interacting with ORC (Origin Recognition Complex) and MCM (Minichromosome Maintenance) proteins. FAD24 (Factor for Adipocyte Differentiation, clone number 24), the human homolog of Noc3p (hNOC3), was previously reported to play roles in the regulation of DNA replication and proliferation in human cells. However, the role of hNOC3 in replication licensing was unclear. Here we report that hNOC3 physically interacts with multiple human pre-replicative complex (pre-RC) proteins and associates with known replication origins throughout the cell cycle. Moreover, knockdown of hNOC3 in HeLa cells abrogates the chromatin association of other pre-RC proteins including hCDC6 and hMCM, leading to DNA replication defects and eventual apoptosis in an abortive S-phase. In comparison, specific inhibition of the ribosome biogenesis pathway by preventing pre-rRNA synthesis, does not lead to any cell cycle or DNA replication defect or apoptosis in the same timeframe as the hNOC3 knockdown experiments. Our findings strongly suggest that hNOC3 plays an essential role in pre-RC formation and the initiation of DNA replication independent of its potential role in ribosome biogenesis in human cells.

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Objectives: The study aimed to examine the anti-diabetic effects of Gynura divaricata (GD) and the underlying mechanism. Methods: Information about the chemical compositions of GD was obtained from extensive literature reports. Potential target genes were predicted using PharmMapper and analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). To validate the results from bioinformatics analyses, an aqueous extract of GD was administered to type 2 diabetic rats established by feeding a high-fat and high-sugar diet followed by STZ injection. Key proteins of the PI3K/AKT signaling pathway and fatty acid metabolism signaling pathway were investigated by immunoblotting. Results: The blood glucose of the rats in the GD treatment group was significantly reduced compared with the model group without treatment. GD also showed activities in reducing the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA). The levels of urine sugar (U-GLU) and urine creatinine (U-CREA) were also lowered after treatment with GD. Bioinformatics analysis showed that some pathways including metabolic pathways, insulin resistance, insulin signaling pathway, PPAR signaling pathway, bile secretion, purine metabolism, etc. may be regulated by GD. Furthermore, GD significantly increased the protein expression levels of PKM1/2, p-AKT, PI3K p85, and GLUT4 in the rat liver. In addition, the expression levels of key proteins in the fatty acid metabolism signaling pathway including AMPK, p-AMPK, PPARα, and CPT1α were significantly upregulated. The antiapoptotic protein BCL-2/BAX expression ratio in rats was significantly upregulated after GD intervention. These results were consistent with the bioinformatics analysis results. Conclusions: Our study suggests that GD can exert hypoglycemic effects in vivo by regulating the genes at the key nodes of the PI3K/AKT signaling pathway and fatty acid metabolism signaling pathway.

Background: Gastric cancer is the leading cause of cancer-related death worldwide. The aim of present study was to investigate the anti-tumor effect of purified Omphalia lapidescens protein (pPeOp) in gastric cancer.Methods: Microarray analysis was performed to find out differentially expressed genes in pPeOp-treated MC-4 gastric cancer cells. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) 3 signaling pathway was most likely to be altered after bioinformatics analysis. Interleukin-6 (IL-6) and NSC74859 were used as the agonist and inhibitor of the JAK/STAT3 signaling pathway, respectively. Flow cytometry and MTS assay were used for cell proliferation and viability analysis in pPeOp-treated gastric cell lines with IL-6 or NSC74859.Results: The anti-tumor effect was increased when pPeOp were co-treated with IL-6, while decreased in inhibitor treatment. The expression of the crucial members in the pathway of MC-4 cells, including glycoprotein 130 (GP130), JAK1, JAK2, STAT3, p-STAT3, suppressor of cytokine signaling SOCS1 and SOCS3, was investigated by western blotting.Conclusion: pPeOp exhibited promising anticancer effect in the xenograft nude mice model, established by STAT3 knock down gastric cancer cells. Thus, JAK/STAT3 inhibition partially contributed to the anticancer effect of pPeOp, which may serve as a novel strategy for gastric cancer.

DNA replication is a highly regulated cellular process in proliferating cells, involving cell cycle dependent assembly of DNA replication-initiation proteins (DRIPs) onto origins of replication. The process of pre-replicative complex (pre-RC) formation at the M-to-G 1 transition, also known as replication licensing, requires origin recognition complex (Orc1-6p) that binds and marks replication origins to facilitate the loading of additional DRIPs, such as Noc3p, Ipi3p, Cdc6p, Cdt1p and Mcm2-7p. The subsequent activation of pre-RC at the G 1 -to-S transition is dependent upon cyclin-dependent kinases (CDKs) and Dbf4-dependent kinase (DDK). This sequential process ensures that DRIPs are precisely loaded to form pre-ICs and then activated by their regulators so that chromosomal DNA is replicated only once per cell cycle. Despite substantial gains in the study of the mechanisms and regulation of pre-RC, the finite details of the pre-RC assembly and disassembly processes remain unclear and controversial. In this review we describe the present state of understanding on DRIPs and the pre-RC architecture and dynamics.

Replication licensing, a prerequisite of DNA replication, helps to ensure once-per-cell-cycle genome duplication. Some DNA replication-initiation proteins are sequentially loaded onto replication origins to form pre-replicative complexes (pre-RCs). ORC and Noc3p bind replication origins throughout the cell cycle, providing a platform for pre-RC assembly. We previously reported that cell cycle–dependent ORC dimerization is essential for the chromatin loading of the symmetric MCM double-hexamers. Here, we usedSaccharomyces cerevisiaeseparation-of-functionNOC3mutants to confirm the separable roles of Noc3p in DNA replication and ribosome biogenesis. We also show that an essential and cell cycle–dependent Noc3p dimerization cycle regulates the ORC dimerization cycle. Noc3p dimerizes at the M-to-G1transition and de-dimerizes in S-phase. The Noc3p dimerization cycle coupled with the ORC dimerization cycle enables replication licensing, protects nascent sister replication origins after replication initiation, and prevents re-replication. This study has revealed a new mechanism of replication licensing and elucidated the molecular mechanism of Noc3p as a mediator of ORC dimerization in pre-RC formation.

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