Reduced expression of GINS complex members induces hallmarks of pre-malignancy in primary untransformed human cells

Abstract

In cancer cells ablation of the GINS complex member Psf2 elicits chromosome mis-segregation yet the precise role of Psf2 in mitosis is unknown. We investigated the putative mitotic role of the GINS complex using synchronized cultures of untransformed Human Dermal Fibroblasts (HDF). Metaphase spreads from Psf1/Psf2-depleted HDF were normal and mitotic exit of Psf1/Psf2-depleted cells was only slightly delayed, suggesting no direct role for the GINS complex in mitosis of untransformed cells. Because the GINS complex is required for initiation and elongation events during DNA replication we hypothesized that the mitotic delay of Psf1/Psf2-deficient cells resulted indirectly from defective DNA synthesis during a prior S-phase. Therefore, we investigated the effects of Psf1/Psf2-depletion on DNA replication. Recruitment of Mcm7 to chromatin during G1 was unaffected by Psf1/Psf2-ablation, indicating that replication licensing does not require GINS. However, chromatin-binding of Cdc45 and PCNA and the onset of DNA synthesis were delayed in Psf1/Psf2-ablated cells. The S-phase delay of Psf1/Psf2-depleted cells was associated with hallmarks of pre-malignancy including acquisition of the DNA damage marker γH2AX, activation of Chk2, and increased Tyr 15 phosphorylation of Cdc2. Ectopic expression of catalytically-inactive Chk2 prevented Cdc2 phosphorylation and resulted in increased numbers of aberrant mitotic cells containing micronuclei, thereby recapitulating the effect of Psf2-deficiency in cancer cells. Therefore, S-phase progression of untransformed cells containing sub-optimal levels of Psf1/2 is associated with replication stress and acquisition of DNA damage. The ensuing Chk2-mediated DNA damage signalling is important for preventing mitotic defects and guarding genome integrity.

A novel role for fanconi anemia (FA) pathway effector protein FANCD2 in cell cycle progression of untransformed primary human cells

“…We used siRNA to target Psf1 and Psf2, components of the GINS complex. As expected from previous studies, 27 GINS-deficiency led to reduced recruitment of the initiation factor Cdc45 to chromatin ( Fig. 3C) and reduced rates of DNA synthesis (Fig.…”

Structure and function of the GINS complex, a key component of the eukaryotic replisome

“…Pre-RC assembly is unaffected in HDF cells, as judged by the loading of MCM on to chromatin, whereas Cdc45 recruitment to chromatin is delayed and the maximal level of chromatin-bound Cdc45 reduced [48]. Further studies suggest that GINS depletion in HeLa human cells leads to mitotic defects also [14], although this is not seen in untransformed HDF cells [48]; this will be discussed further shortly.…”

“…Transfection of siRNAs directed against the GINS subunits individually into HeLa cells reduces GINS levels to <10 % of that seen in control transfected cells and inhibiting the expression of any one subunit of the complex results in degradation of the others [45]. Cell proliferation and DNA replication (measured by uptake of labelled precursors and flow cytometry) is significantly impaired in HeLa and HDF (human dermal fibroblast) cells transfected with GINS-targeted siRNAs, with defects seen at both the initiation and elongation stages of replication and the apparent accumulation of DNA damage [45,48]. Pre-RC assembly is unaffected in HDF cells, as judged by the loading of MCM on to chromatin, whereas Cdc45 recruitment to chromatin is delayed and the maximal level of chromatin-bound Cdc45 reduced [48].…”

“…Cell proliferation and DNA replication (measured by uptake of labelled precursors and flow cytometry) is significantly impaired in HeLa and HDF (human dermal fibroblast) cells transfected with GINS-targeted siRNAs, with defects seen at both the initiation and elongation stages of replication and the apparent accumulation of DNA damage [45,48]. Pre-RC assembly is unaffected in HDF cells, as judged by the loading of MCM on to chromatin, whereas Cdc45 recruitment to chromatin is delayed and the maximal level of chromatin-bound Cdc45 reduced [48]. Further studies suggest that GINS depletion in HeLa human cells leads to mitotic defects also [14], although this is not seen in untransformed HDF cells [48]; this will be discussed further shortly.…”

PSF1, a DNA Replication Factor Expressed Widely in Stem and Progenitor Cells, Drives Tumorigenic and Metastatic Properties

“…In yeast, the GINS complex associates with the MCM2-7 complex and CDC45, and this "C-M-G complex" (CDC45-MCM2-7-GINS) regulates both the initiation and the progression of DNA replication (1)(2)(3)(4)(5)(6). In Xenopus and human, GINS has been suggested to be involved in DNA replication because of its binding to DNA replication protein (7)(8)(9)(10); however, a recent study suggests that PSF1/2 is associated with the response to replication stress and acquisition of DNA damage in untransformed human dermal fibroblasts (11). Thus, the exact functions of GINS components in mammalian cells are not yet clear.…”