Retardation of cell cycle progression in yeast cells recovering from DNA damage: A study at the single cell level

Abstract

Pedigree analyses of individual yeast cells recovering from DNA damage were performed and time intervals between morphological landmark events during the cell cycle (bud emergence and cell separation), were recorded for three generations. The associated nuclear behavior was monitored with the aid of DAPI staining. The following observations were made: All agents tested (X-rays, MMS, EMS, MNNG, nitrous acid) delayed the first bud emergence after treatment, which indicates inhibition of the initiation of DNA replication. Cells that survived X-irradiation progressed further through the cell cycle in a similar way to control cells. Progress of chemically treated cells became extremely asynchronous because surviving cells stayed undivided for periods of varying length. Prolongation of the time between bud emergence and cell separation was most pronounced for cells treated with the alkylating agents MMS and EMS. This is interpreted as retardation of ongoing DNA synthesis by persisting DNA adducts. Cell cycle prolongation in the second and third generation after treatment was observed only with MMS treated cells. In all experiments, individual cells of uniformly treated populations exhibited highly variable responses.

Masking and purging mutations following EMS treatment in haploid, diploid and tetraploid yeast (Saccharomyces cerevisiae)

“…It has been suggested that delay of bud emergence after DNA damage may be due to active inhibition of DNA replication during periods of extensive DNA repair (Wintersberger & Karwan, 1987). In our experiments, prototrophic diploid cells treated with 50 µl EMS first showed evidence of normal budding after 14 h of growth whereas normal budding of treated haploid cells was not apparent until after transfer to fresh YPD medium i.e.…”

“…We chose the mutagen EMS for study because it has been extensively studied in yeast (Loprieno, 1966 ;Nasim & Auerbach, 1967 ;Prakash & Sherman, 1973 ;Prakash & Higgins, 1982 ; see also Kilbey & Hunter, 1983 ;Schiestl & Wintersberger, 1983 ;van Zeeland et al, 1983 ;Orthen et al, 1984 ;e.g. reviewed by Sega, 1984 ;Wintersberger & Karwan, 1987 ;Klein et al, 1989 ;Klein et al, 1990 ;Lee et al, 1992). EMS is an alkylating agent that causes DNA damage ; repair of this damage induces a high frequency of G\C to A\T transition mutations in yeast (Prakash & Sherman, 1973 ;Sega, 1984 ;Kohalmi & Kunz, 1988 ;Lee et al, 1992).…”

Requirement for End-Joining and Checkpoint Functions, but NotRAD52-Mediated Recombination, after EcoRI Endonuclease Cleavage of Saccharomyces cerevisiaeDNA

“…Exposure of yeast cells to ionizing radiation or methyl methanesulfonate causes growing cells to arrest in G 2 . Arrested cells retain large buds and a single undivided nucleus (55,71,80,82). Initial experiments established that rad9 strains were deficient in DNA damageinduced cell cycle arrest (80), and additional genes were subsequently identified (71).…”

RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae.

“…The cessation of DNA replication triggered by DNA damage has been demonstrated in Escherichia coli (3). Additionally, several studies indicate that a variety of DNA-damaging agents inhibit DNA replication in yeast (4). However, it is not known whether this response requires an active regulatory process or is exclusively the result of the passive stalling of replicative and/or transcriptional complexes at sites of base damage.…”