Article

Cell cycle-dependent phosphorylation of Rad53 kinase by Cdc5 and Cdc28 modulates checkpoint adaptation.

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
Cell cycle (Georgetown, Tex.) (Impact Factor: 5.01). 01/2010; 9(2):350-63. DOI: 10.4161/cc.9.2.10448
Source: PubMed

ABSTRACT In budding yeast the evolutionarily conserved checkpoint response varies in its sensitivity to DNA damaging agents through the cell cycle. Specifically, higher amounts of damage are needed to activate the downstream checkpoint kinase Rad53 in S-phase cells. We examined here whether phosphorylation of Rad53 itself by cell cycle-dedicated kinases regulates Rad53 activation. We found that during unperturbed growth Rad53 exhibits a small phosphorylation-dependent electrophoretic mobility shift in G(2), M and G(1) phases of the cell cycle that is lost in S phase. We show that Rad53 is phosphorylated in vitro by Cdc5, a mitotic Polo-like kinase, and by the yeast cyclin-dependent kinase, Cdc28. Consistently, the cell cycle-dependent Rad53 mobility shift requires both Cdc5 and Cdc28 activities. We mapped the in vitro targeted phosphorylation sites by mass spectrometry and confirmed with mass spectroscopy that serines 774, 789 and 791 within Rad53 are phosphorylated in vivo in M-phase arrested cells. By creating nonphosphorylatable mutations in the endogenous RAD53 gene, we confirmed that the CDK and Polo kinase target sites are responsible for the observed cell cycle-dependent shift in protein mobility. The loss of phospho-acceptor sites does not interfere with Rad53 activation but accelerates checkpoint adaptation after induction of a single irreparable double-strand break. We thus demonstrate that cell cycle-dependent phosphorylation can fine-tune the response of Rad53 to DNA damage.

1 Follower
 · 
165 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The drive to proliferate and the need to maintain genome integrity are two of the most powerful forces acting on biological systems. When these forces enter in conflict, such as in the case of cells experiencing DNA damage, feedback mechanisms are activated to ensure that cellular proliferation is stopped and no further damage is introduced while cells repair their chromosomal lesions. In this circumstance, the DNA damage response dominates over the biological drive to proliferate, and may even result in programmed cell death if the damage cannot be repaired efficiently. Interestingly, the drive to proliferate can under specific conditions overcome the DNA damage response and lead to a reactivation of the proliferative program in checkpoint-arrested cells. This phenomenon is known as adaptation to DNA damage and is observed in all eukaryotic species where the process has been studied, including normal and cancer cells in humans. Polo-like kinases (PLKs) are critical regulators of the adaptation response to DNA damage and they play key roles at the interface of cell cycle and checkpoint-related decisions in cells. Here, we review recent progress in defining the specific roles of PLKs in the adaptation process and how this conserved family of eukaryotic kinases can integrate the fundamental need to preserve genomic integrity with effective cellular proliferation.
    Systems and Synthetic Biology 09/2014; 8(3). DOI:10.1007/s11693-014-9151-9
  • [Show abstract] [Hide abstract]
    ABSTRACT: Unlike most conventional techniques with static model assumption, this paper aims to estimate the time-varying model parameters and identify significant genes involved at different timepoints from time course gene microarray data. We first formulate the parameter identification problem as a new maximum a posteriori probability estimation problem so that prior information can be incorporated as regularization terms to reduce the large estimation variance of the high dimensional estimation problem. Under this framework, sparsity and temporal consistency of the model parameters are imposed using L1-regularization and novel continuity constraints, respectively. The resulting problem is solved using the L-BFGS method with the initial guess obtained from the partial least squares method. A novel forward validation measure is also proposed for the selection of regularization parameters, based on both forward and current prediction errors. The proposed method is evaluated using a synthetic benchmark testing data and a publicly available yeast Saccharomyces cerevisiae cell cycle microarray data. For the latter particularly, a number of significant genes identified at different timepoints are found to be biological significant according to previous findings in biological experiments. These suggest that the proposed approach may serve as a valuable tool for inferring time-varying gene regulatory networks in biological studies.
    IEEE/ACM Transactions on Computational Biology and Bioinformatics 01/2015; 12(1):123-135. DOI:10.1109/TCBB.2014.2343951 · 1.54 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Checkpoints monitor critical cell cycle events such as chromosome duplication and segregation. They are highly conserved mechanisms that prevent progression into the next phase of the cell cycle when cells are unable to accomplish the previous event properly. During S phase, cells also provide a surveillance mechanism called the DNA replication checkpoint, which consists of a conserved kinase cascade that is provoked by insults that block or slow down replication forks. The DNA replication checkpoint is crucial for maintaining genome stability, because replication forks become vulnerable to collapse when they encounter obstacles such as nucleotide adducts, nicks, RNA-DNA hybrids, or stable protein-DNA complexes. These can be exogenously induced or can arise from endogenous cellular activity. Here, we summarize the initiation and transduction of the replication checkpoint as well as its targets, which coordinate cell cycle events and DNA replication fork stability.
    09/2013; 4(3):388-434. DOI:10.3390/genes4030388

Full-text

Download
9 Downloads
Available from
Oct 21, 2014
Available from