PubMed · 8608021
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1995. Cell multiplication.. https://doi.org/10.1016/0955-0674(95)80076-x
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Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.
The RAD6 pathway is central to post-replicative DNA repair in eukaryotic cells; however, the machinery and its regulation remain poorly understood. Two principal elements of this pathway are the ubiquitin-conjugating enzymes RAD6 and the MMS2-UBC13 heterodimer, which are recruited to chromatin by the RING-finger proteins RAD18 and RAD5, respectively. Here we show that UBC9, a small ubiquitin-related modifier (SUMO)-conjugating enzyme, is also affiliated with this pathway and that proliferating cell nuclear antigen (PCNA) -- a DNA-polymerase sliding clamp involved in DNA synthesis and repair -- is a substrate. PCNA is mono-ubiquitinated through RAD6 and RAD18, modified by lysine-63-linked multi-ubiquitination--which additionally requires MMS2, UBC13 and RAD5--and is conjugated to SUMO by UBC9. All three modifications affect the same lysine residue of PCNA, suggesting that they label PCNA for alternative functions. We demonstrate that these modifications differentially affect resistance to DNA damage, and that damage-induced PCNA ubiquitination is elementary for DNA repair and occurs at the same conserved residue in yeast and humans.
Type I and type II interferons (IFNs) are known to exert antitumor effects on a variety of tissues and cell types. We have previously shown that the type I IFN IFN alpha induces the expression of the cyclin-dependent kinase inhibitor p21WAF1 and inhibits the cell cycle of the human prostate adenocarcinoma cell line, DU145, that carries mutations in the tumor suppressor gene products p53 and pRB. We now show that the type II IFN IFN gamma similarly induces the expression of p21WAF1 and inhibits the cell cycle of DU145 cells. In addition, we show that while both IFNs exert antiproliferative activity, only IFN gamma induced phenotypic changes in these cells that accompanied the antiproliferative effect. For example, IFN gamma, but not IFN alpha, caused a significant reduction in epidermal growth factor receptor expression as well as an increase in the adhesion molecules intercellular adhesion molecule-1 and integrin alpha3. These phenotypic changes in DU145 cells are suggestive of the acquisition of a non-tumorigenic state. Consistent with these findings, IFN gamma showed a significantly lower invasive ability in in vitro assays using invasion chambers. Thus, IFN gamma inhibits both the cell cycle and the metastatic potential of DU145 cells independent of the p53 and RB status, and our data describe a mechanism for mediating the antitumor capabilities of IFN gamma that bypasses tumor suppressor genes like p53.