DNA replication is a stringently regulated cellular process. In proliferating cells, DNA replication-initiation proteins (RIPs) are sequentially loaded onto replication origins during the M-to-G₁ transition to form the pre-replicative complex (pre-RC), a process known as replication licensing. Subsequently, additional RIPs are recruited to form the pre-initiation complex (pre-IC). RIPs and their regulators ensure that chromosomal DNA is replicated exactly once per cell cycle. Origin recognition complex (ORC) binds to, and marks replication origins throughout the cell cycle and recruits other RIPs including Noc3p, Ipi1-3p, Cdt1p, Cdc6p and Mcm2-7p to form the pre-RC. The detailed mechanisms and regulation of the pre-RC and its exact architecture still remain unclear. In this study, pairwise protein-protein interactions among 23 budding yeast and 16 human RIPs were systematically and comprehensively examined by yeast two-hybrid analysis. This study tested 470 pairs of yeast and 196 pairs of human RIPs, from which 113 and 96 positive interactions, respectively, were identified. While many of these interactions were previously reported, some were novel, including various ORC and MCM subunit interactions, ORC self-interactions, and the interactions of IPI3 and NOC3 with several pre-RC and pre-IC proteins. Ten of the novel interactions were further confirmed by co-immunoprecipitation assays. Furthermore, we identified the conserved interaction networks between the yeast and human RIPs. This study provides a foundation and framework for further understanding the architectures, interactions and functions of the yeast and human pre-RC and pre-IC.

Backgrounds: Non-alcoholic fatty liver disease (NAFLD) is a multifaceted metabolic disorder that has become a prominent public health problem worldwide. As a traditional Chinese medicine formula, the ErChen decoction (ECD) possesses significant effects on metabolic syndrome. Methods: To determine whether ECD can relieve lipid accumulation and insulin resistance (IR) in liver cells, NAFLD and IR cell models were established by treating HepG2 cells with free fatty acids and an overdose of insulin, respectively. Bioinformatics and experimental evidence demonstrated that ECD could ameliorate NAFLD by modulating multiple pathways. The optimal combination of the key compounds in ECD was identified by the orthogonal experiment. Results: For lipid homeostasis, ECD suppressed de novo lipogenesis and reduced the cholesterol level by activating the AMPK signaling pathway. Concurrently, ECD enhanced hepatic β-oxidation by inducing PPARα-mediated upregulation of ACOX-1 and CPT-1α. ECD also resolved hepatic insulin resistance by activating the IRS1-Akt-FoxO1 pathway. The combined treatment with 100 μM liquiritin (LQ), 200 μM glycyrrhizic acid (GA) and 200 μM hesperidin (HEN) exhibited the best effect in reducing TG content in NAFLD model cells. Conclusions: ECD exhibited superior activities in activating the AMPK signaling pathway compared to the optimal compound combination. The comparison between the ECD and its key compounds demonstrated the superior synergistic effects of the herbs in ECD.

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-G₁ 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.

Primary cilia are microtubule-based, antenna-like organelles, which are formed in G ₀ phase and resorbed as cells re-enter the cell cycle. It has been reported that the length of primary cilia can influence the timing of cell cycle progression. However, the molecular links between ciliogenesis and cell cycle progression are not clear. FOP (Fibroblast growth factor receptor 1 Oncogene Partner, also known as FGFR1OP) has been implicated in ciliogenesis. Here, we show that the expression of FOP during cell cycle exit and re-entry is negatively correlated with ciliogenesis. Knockdown of FOP promotes cilia elongation and suppresses timely cilia disassembly. In contrast, ectopic expression of FOP inhibits cilia growth. Moreover, pharmacological inhibition of actin polymerization with Cytochalasin D abrogates FOP-induced cilia disassembly, suggesting that FOP facilitates cilia disassembly by promoting actin cytoskeleton formation. Lastly, knockdown of FOP delays cell cycle re-entry of quiescent cells following serum re-stimulation, and this can be reversed by silencing IFT20 (intraflagellar transport 20), an intraflagellar transport protein essential for ciliogenesis. Collectively, these results suggest that FOP plays a negative role in ciliogenesis and can promote cell cycle re-entry by facilitating cilia disassembly.

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.