A Role of hIPI3 in DNA Replication Licensing in Human Cells

Abstract

The yeast Ipi3p is required for DNA replication and cell viability in Sacharomyces cerevisiae. It is an essential component of the Rix1 complex (Rix1p/Ipi2p-Ipi1p-Ipi3p) that is required for the processing of 35S pre-rRNA in pre-60S ribosomal particles and for the initiation of DNA replication. The human IPI3 homolog is WDR18 (WD repeat domain 18), which shares significant homology with yIpi3p. Here we report that knockdown of hIPI3 resulted in substantial defects in the chromatin association of the MCM complex, DNA replication, cell cycle progression and cell proliferation. Importantly, hIPI3 silencing did not result in a reduction of the protein level of hCDC6, hMCM7, or the ectopically expressed GFP protein, indicating that protein synthesis was not defective in the same time frame of the DNA replication and cell cycle defects. Furthermore, the mRNA and protein levels of hIPI3 fluctuate in the cell cycle, with the highest levels from M phase to early G1 phase, similar to other pre-replicative (pre-RC) proteins. Moreover, hIPI3 interacts with other replication-initiation proteins, co-localizes with hMCM7 in the nucleus, and is important for the nuclear localization of hMCM7. We also found that hIPI3 preferentially binds to the origins of DNA replication including those at the c-Myc, Lamin-B2 and β-Globin loci. These results indicate that hIPI3 is involved in human DNA replication licensing independent of its role in ribosome biogenesis.

The interaction networks of the budding yeast and human DNA replication-initiation proteins

“…Comparatively, both yeast and human IPI3 interacted with ORC2 and MCM4 [9,28]. The collective evidence is consistent with human IPI3, being a licensing factor in pre-RC formation in both yeast and human cells.…”

“…We have also previously reported that yIpi3p interacts with yOrc2p, yCdc6p, yCdt1p and yMcm4 [9] (Supplementary Figure S3 and Supplementary Table S1). We also reported that hIPI3 (WDR18) interacts with hORC1, hORC2, hORC3, hORC4, hORC5, hMCM2, hMCM3, hMCM4 and hMCM7 [28].…”

“…The self-interactions of ORC subunits are also of biological significance (unpublished). The interactions of Noc3p and Ipi3 with pre-RC and pre-IC components further suggest that these proteins are involved in DNA replication as well as ribosome biogenesis [9,13,28]. In addition, the biological significance of the interactions among GINS, ORC, Cdt1 and Ipi3 proteins are worth further study.…”

An Essential and Cell-Cycle-Dependent ORC Dimerization Cycle Regulates Eukaryotic Chromosomal DNA Replication

“…In addition to the inter-subunit interactions that we and others previously reported (Matsuda et al, 2007;Chen et al, 2008;Huo et al, 2012;Wu et al, 2019), we found that Orc1p, Orc2p, Orc5p, and Orc6p have self-interactions, i.e., molecules of the same subunit interact with each other ( Figure 1A). Of note, yeast two-hybrid analysis based on the relative yeast growth rates on different selection stringencies, as we used in this study, favors the detection of direct physical interactions, most of which can be confirmed by other methods (Yu et al, 2004;Huo et al, 2012;Huang et al, 2016;Cheung et al, 2019;Wu et al, 2019; this study).…”

Human NOC3 is essential for DNA replication licensing in human cells

“…In budding yeast, the six subunits of ORC and Noc3p bind to replication origins throughout the cell cycle, where they function as the origin recognition scaffold [3][4][5]. Pre-RC assembly begins at the M-to-G 1 transition with the loading of Cdc6p by Orc1-6p, Noc3p and Ipi3 [3][4][5][6], followed by the association of the Cdt1p-MCM2-7p heptamer [7][8][9][10][11][12][13][14][15][16]. After pre-RC assembly, replication origins are "licensed" and ready for firing (origin activation) [17][18][19].…”