A Mammalian-Like DNA Damage Response of Fission Yeast to Nucleoside Analogs

Abstract

Nucleoside analogs are frequently used to label newly synthesized DNA. These analogs are toxic in many cells, with the exception of the budding yeast. We show that Schizosaccharomyces pombe behaves similarly to metazoans in response to analogs 5-bromo-29-deoxyuridine (BrdU) and 5-ethynyl-29-deoxyuridine (EdU). Incorporation causes DNA damage that activates the damage checkpoint kinase Chk1 and sensitizes cells to UV light and other DNA-damaging drugs. Replication checkpoint mutant cds1Δ shows increased DNA damage response after exposure. Finally, we demonstrate that the response to BrdU is influenced by the ribonucleotide reductase inhibitor, Spd1, suggesting that BrdU causes dNTP pool imbalance in fission yeast, as in metazoans. Consistent with this, we show that excess thymidine induces G1 arrest in wild-type fission yeast expressing thymidine kinase. Thus, fission yeast responds to nucleoside analogs similarly to mammalian cells, which has implications for their use in replication and damage research, as well as for dNTP metabolism.

Cell-Cycle Analyses Using Thymidine Analogues in Fission Yeast

“…4A). Consistently, Sabatinos et al observed a more severe effect on the second S phase than on the first one after release from a HU block in the presence of EdU [16]. We speculate that the cells may have problems replicating the EdU-labelled DNA and thus the DNA-damage checkpoint might be activated in the second cell cycle.…”

“…We conclude that the detrimental effects of the analogues can not be solely explained by incorporation into the DNA. Consistently, BrdU has been shown to affect the cell-cycle progression by a mechanism not related to its incorporation into the chromosomal DNA [16]. With increasing BrdU-concentrations, the effects on cell-cycle progression became more severe, even when the amount of BrdU incorporated into the DNA was saturated [16].…”

Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S, and G2 Phases of the Cell Cycle in Trypanosomatids

“…Thus, in trypanosomatids, the imbalance of the nucleotide pool caused by treatments with different concentrations of thymidine analogs for 60 min was apparently insufficient to cause a feedback inhibition in the synthesis of nucleotides because after the addition of different concentrations of BrdU, none of the three trypanosomatids analyzed showed significant changes in the DNA content profile that could be interpreted as cell cycle arrest (Fig. S2), as demonstrated in mammals and yeast (Hyland et al 2000;Popescu 1999;Sabatinos et al 2013). In conclusion, a greater efficiency of DNA denaturation by increasing the HCl concentration to 3 M can eradicate the differences observed in DNA monitoring, strongly suggesting that the discrepancies identified are due to ineffective detection of BrdU and not to inefficient incorporation.…”

“…; Popescu ; Sabatinos et al. ). In conclusion, a greater efficiency of DNA denaturation by increasing the HCl concentration to 3 M can eradicate the differences observed in DNA monitoring, strongly suggesting that the discrepancies identified are due to ineffective detection of BrdU and not to inefficient incorporation.…”

Importancia de la fase G1 del ciclo celular en medios pobres en nitrógeno en Schizosaccharomyces pombe

“…En respuesta a diferentes lesiones, como roturas de la doble cadena del DNA o a horquillas colapsadas, Rad52 permite la carga de proteínas de recombinación en el DNA generando focos de recombinación (Muris et al, 1997) que suelen aparecer al final de la fase S o al inicio de la fase G2. En un cultivo asíncrono de la cepa silvestre de S. pombe sólo el 1% de las células presentan más de un foco de Rad52-YFP y el 10-14% de las células presentan un foco que corresponde a la reparación del DNA tras la replicación (Meister et al, 2005;Bailis et al, 2008;Sabatinos et al, 2013).…”