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.
Highlights
In eukaryotes, the cell cycle can be divided in two major stages: the first is interphase, which is for cell growth, genome duplication and preparation for mitosis, and the second is mitotic phase in which the cell separates itself into two cells
Silencing of hIPI3 resulted in substantial defects in the chromatin association of MCM proteins, DNA replication, cell cycle progression and cell proliferation in human cells
Since all pre-replicative complex (pre-RC) proteins are normally present on chromatin at the G1/S boundary in untreated cells without hIPI3 silencing, these results suggest that hIPI3 is involved in replication licensing upstream of hCDC6 and MCM proteins and downstream of hORC and hNOC3, reminiscent of the findings in budding yeast [4]
Summary
The cell cycle can be divided in two major stages: the first is interphase, which is for cell growth, genome duplication and preparation for mitosis, and the second is mitotic phase in which the cell separates itself into two cells. It must replicate its genomic DNA so that the two daughter cells have the same genetic information as their parent. The replicators and origins of replication are special sequences in the genome, serving as the binding sites for replication-initiation proteins and the start sites, respectively, for the initiation of DNA replication [1, 2]. The replication origins are usually AT rich sequences which are more unwound [3].
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