Abstract
Apoptosis in metazoans is often accompanied by the destruction of DNA replication initiation proteins, inactivation of checkpoints and activation of cyclin-dependent kinases, which are inhibited by checkpoints that directly or indirectly require initiation proteins. Here we show that, in the budding yeast Saccharomyces cerevisiae, mutations in initiation proteins that attenuate both the initiation of DNA replication and checkpoints also induce features of apoptosis similar to those observed in metazoans. The apoptosis-like phenotype of initiation mutants includes the production of reactive oxygen species (ROS) and activation of the budding-yeast metacaspase Yca1p. In contrast to a recent report that activation of Yca1p only occurs in lysed cells and does not contribute to cell death, we found that, in at least one initiation mutant, Yca1p activation occurs at an early stage of cell death (before cell lysis) and contributes to the lethal effects of the mutation harbored by this strain. Apoptosis in initiation mutants is probably caused by DNA damage associated with the combined effects of insufficient DNA replication forks to completely replicate the genome and defective checkpoints that depend on initiation proteins and/or replication forks to restrain subsequent cell-cycle events until DNA replication is complete. A similar mechanism might underlie the proapoptotic effects associated with the destruction of initiation and checkpoint proteins during apoptosis in mammals, as well as genome instability in initiation mutants of budding yeast.
Highlights
DNA replication is coupled to the celldivision cycle by the reciprocal regulation of proteins required for initiation at origins of DNA replication and molecular events regulated by cyclin-dependent kinases (CDKs) that drive the cell cycle forward
In G1 and early S phase cells, CDKs and/or CDK-dependent molecular events required for completion of S phase and mitosis are, in turn, inhibited by checkpoints that depend on pre-RC proteins, either directly (Clay-Farrace et al, 2003; Oehlmann et al, 2004; Toyn et al, 1995; Weinreich et al, 2001) or indirectly through the establishment of DNA replication forks (Tercero et al, 2003)
Parallel assessments of cell death detected by membrane changes that allow for staining of DNA in dead or dying cells by propidium iodide, a membrane-impermeable dye, indicated that both reactive oxygen species (ROS) production and cell death were rapid, and suggested that ROS production can precede loss of viability (Fig. 1C)
Summary
DNA replication is coupled to the celldivision cycle by the reciprocal regulation of proteins required for initiation at origins of DNA replication and molecular events regulated by cyclin-dependent kinases (CDKs) that drive the cell cycle forward. Apoptosis coincides with the destruction of the pre-RC proteins Cdc (Blanchard et al, 2002; Burhans et al, 2002; Illenye and Heintz, 2004; Pelizon et al, 2002; Schories et al, 2004; Yim et al, 2003; Yin et al, 2004) and Mcm (Schwab et al, 1998), the CDK inhibitors Wee (Zhou et al, 1998), p21 and p27 (Levkau et al, 1998) (for a review, see Gartel and Tyner, 2002), and the checkpoint proteins ATM (Smith et al, 1999), Rb (Tan et al, 1997) and Rad (Lee et al, 2003). In addition to the attenuation of apoptosis that occurs when destruction of CDK inhibitors are blocked, apoptosis is attenuated when CDK activation is directly inhibited by, 3544 Journal of Cell Science 118 (15)
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