PubMed · 7949418
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P Nurse. 1994. Fission yeast morphogenesis--posing the problems.. https://doi.org/10.1091/mbc.5.6.613
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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.
p27(Kip1) regulates the decision to enter into S-phase or withdraw from the cell cycle by establishing an inhibitory threshold above which G1 cyclin-dependent kinases accumulate before activation. We have used the HL-60 cell line to study regulation of p27 as cells withdraw from the cell cycle following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We found that the amount of p27 is maximal in G0 cells, lower in G1 cells, and undetectable in S-phase cells. In contrast to the protein, the amount of p27 mRNA was the same in these populations, suggesting that accumulation of p27 during the cell cycle and as cells withdraw from the cell cycle is controlled by post-transcriptional mechanisms. In S-phase cells, the degradation of p27 appears to predominate as a regulatory mechanism. In G0 cells, there was an increase in the synthesis rate of p27. Our data demonstrate that, in G0 cells, accumulation of p27 is due to an increase in the amount of p27 mRNA in polyribosomes.