The DNA damage checkpoint: A tale from budding yeast

Abstract

Studies performed in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have led the way in defining the DNA damage checkpoint and in identifying most of the proteins involved in this regulatory network, which turned out to have structural and functional equivalents in humans. Subsequent experiments revealed that the checkpoint is an elaborate signal transduction pathway that has the ability to sense and signal the presence of damaged DNA and transduce this information to influence a multifaceted cellular response that is essential for cancer avoidance. This review focuses on the work that was done in Saccharomyces cerevisiae to articulate the checkpoint concept, to identify its players and the mechanisms of activation and deactivation.

Keywords: DNA damage; cell cycle; checkpoint; protein kinases; yeast.

FIGURE 1

Budding yeast mitotic cell cycle. During the G1 phase, cells are unbudded and contain a single spindle pole body (SPB) (red square). Once cells become committed to enter S phase, they start budding, duplicate the SPB and replicate DNA (blue lines). In G2, the mitotic spindle (green lines) is assembled along the mother-daughter axis and the nucleus (light blue circle) moves to the bud neck. In M phase, duplicated chromosomes get attached (yellow dots) to the microtubules in a bipolar manner in metaphase and are pulled apart during anaphase. Once chromosome segregation is completed (telophase), the spindle disassembles and the 2 cells get physically separated. The nuclear envelope does not breakdown. Only one chromosome is shown. DNA damage in G1 activates a checkpoint that arrests the G1/S transition, whereas DNA damage in G2 after completion of DNA replication activates a checkpoint that arrests the metaphase to anaphase transition. Detection of DNA lesions during S phase elicits a checkpoint response that controls completion of DNA replication before cells enter M phase.

FIGURE 2

Simplified DNA damage checkpoint architecture in S. cerevisiae, S. pombe and H. sapiens. See text for details.

FIGURE 3

Model for Rad53 activation in response to DNA DSBs. The MRX-Sae2 complex is rapidly recruited to DNA ends. Rad9 is already bound to chromatin via interaction with methylated histone H3 (yellow dots). MRX bound to DNA ends recruits and activates Tel1, which in turn phosphorylates histone H2A on S129 (green dots), an event that leads to a further enrichment of Rad9 at DSBs. DSB end processing by Exo1 and Dna2-Sgs1 nucleases generates ssDNA that is coated by RPA. RPA-coated ssDNA allows the recruitment of Mec1-Ddc2 and a switch from Tel1 to Mec1 signaling. The 9-1-1 clamp loader recruits the 9-1-1 complex at the 5′ recessed end of the ssDNA-dsDNA junction. Mec1 in turn phosphorylates the Ddc1 subunit of the 9-1-1 complex (green dots), thus creating a docking site for Dpb11 binding. Rad9, once phosphorylated by Cdk1 (white dots), can also bind to Dpb11 that acts as a scaffold to promote Rad9-Mec1 interaction and therefore Rad9 phosphorylation by Mec1. Phosphorylated Rad9 first acts as an adaptor to bring Rad53 into close proximity to Mec1 to allow Mec1-dependent Rad53 phosphorylation. Then, Rad9 promotes Rad53 in trans-autophosphorylation (light blue dots) by increasing the local concentration of Rad53 molecules. Fully activated Rad53 molecules are then released from the Rad9 complex. Rad9 oligomerization is not shown.