Biomedical subjects

David P Toczyski

Publications and source records attributed to David P Toczyski.

10 recordsLinked to original sources

PubMed2006

Precise destruction: an emerging picture of the APC.

Cell cycle transitions are often accompanied by the degradation of regulatory molecules. Targeting proteins to the proteasome for degradation is accomplished by the covalent addition of ubiquitin chains. The specificity of this pathway is largely dictated by a set of enzymes called ubiquitin ligases (or E3s). The anaphase-promoting complex (or APC) is a ubiquitin ligase that has a particularly prominent role in regulating cell cycle progression. To date, the APC is the most complicated member of the RING/cullin family of multisubunit E3s. It includes at least 13 core subunits and three related adaptors. A combination of biochemical, genetic, and structural approaches are now shedding light on the enzymology of the APC. This review will focus on these data, drawing parallels with related ubiquitin ligases.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2006

Taking it off: regulation of H3 K56 acetylation by Hst3 and Hst4.

Histone modifications have been implicated in both DNA repair and checkpoint-mediated responses to DNA damage. Recently much attention has focused on the acetylation of H3 K56. Indeed, this modification is cell cycle-regulated, maintained upon replicative damage in a checkpoint-dependent manner, and is essential for surviving DNA damage. We and others have discovered that two members of the HDAC Sirtuin family, Hst3 and Hst4, negatively regulate H3 K56 acetylation in budding yeast. Additionally, we have shown that these two HDACs are targeted for repression by the DNA damage checkpoint, which is vital for DNA damage tolerance. Discovery that two HDACs are negative regulators of the cellular response to DNA damage and that they target the acetylation of H3 K56 reveals a complex relationship between histone modifications, HDACs, and the DNA damage response. Here, we discuss the recent reports of the regulation of H3 K56-Ac by Hst3 and Hst4 and put forth the critical questions that remain for understanding the intimate, though poorly characterized, connection between chromatin states and genomic maintenance.

Acetylation↗

PubMed2006

Cell cycle and checkpoint regulation of histone H3 K56 acetylation by Hst3 and Hst4.

Histone modifications, including H3 K56 acetylation, have been implicated in DNA damage tolerance. Here, we present evidence that Hst3 and Hst4, two paralogues of the histone deacetylase Sir2, target the cell cycle-regulated acetylation of H3 on K56 and are downregulated during DNA damage in a checkpoint-dependent manner. We show that Hst3 and Hst4 are themselves cell cycle regulated and that their misexpression affects H3 K56-Ac. Moreover, a histone H3 K56R mutation is epistatic to all phenotypes caused by HST3/4 deletion or HST3 overexpression, suggesting that H3K56-Ac is the major target of these histone deacetylases. On examining 18 members of the "Clb2 cluster" of cell cycle-regulated proteins to which Hst3 belongs, we find that two others, Ynl058c and Alk1, are significantly downregulated on DNA damage. Taken together, our data show that Hst3/Hst4 are negative regulators of H3 K56-Ac and that HST3 downregulation by a checkpoint-mediated transcriptional repression system is essential for surviving DNA damage.

Acetylation↗

PubMed2006

An architectural map of the anaphase-promoting complex.

The anaphase-promoting complex or cyclosome (APC) is an unusually complicated ubiquitin ligase, composed of 13 core subunits and either of two loosely associated regulatory subunits, Cdc20 and Cdh1. We analyzed the architecture of the APC using a recently constructed budding yeast strain that is viable in the absence of normally essential APC subunits. We found that the largest subunit, Apc1, serves as a scaffold that associates independently with two separable subcomplexes, one that contains Apc2 (Cullin), Apc11 (RING), and Doc1/Apc10, and another that contains the three TPR subunits (Cdc27, Cdc16, and Cdc23). We found that the three TPR subunits display a sequential binding dependency, with Cdc27 the most peripheral, Cdc23 the most internal, and Cdc16 between. Apc4, Apc5, Cdc23, and Apc1 associate interdependently, such that loss of any one subunit greatly reduces binding between the remaining three. Intriguingly, the cullin and TPR subunits both contribute to the binding of Cdh1 to the APC. Enzymatic assays performed with APC purified from strains lacking each of the essential subunits revealed that only cdc27Delta complexes retain detectable activity in the presence of Cdh1. This residual activity depends on the C-box domain of Cdh1, but not on the C-terminal IR domain, suggesting that the C-box mediates a productive interaction with an APC subunit other than Cdc27. We have also found that the IR domain of Cdc20 is dispensable for viability, suggesting that Cdc20 can activate the APC through another domain. We have provided an updated model for the subunit architecture of the APC.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2006

Methods for studying adaptation to the DNA damage checkpoint in yeast.

When yeast are faced with irreparable DNA damage, they will first arrest in G2/M, via the DNA damage checkpoint pathway, but will subsequently adapt to that arrest and resume division. Here, we summarize assays that we have used to examine checkpoint adaptation. Specifically, we discuss the merits of inducing DNA damage with ionizing radiation (IR) and IR-mimetic drugs, HO, and the cdc13-1 mutation. We also discuss readouts that we have used to visualize adaptation.

Cell Cycle↗

PubMed2005

Complicated tails: histone modifications and the DNA damage response.

In recent years, several ATP-dependent chromatin-remodeling complexes and covalent histone modifications have been implicated in the response to double-stranded DNA breaks (DSBs). When a DSB occurs, cells must identify the DSB, activate the DNA damage checkpoint, and repair the break. Chromatin modification appears to be important but not essential for each of these processes, yet its precise mechanistic roles are only beginning to come into focus. Here, we discuss the role of chromatin in signaling by the DNA damage checkpoint pathway.

Animals↗

PubMed2005

Damage in transition.

Double-stranded DNA breaks (DSBs) are a particularly dangerous form of DNA damage because they can lead to chromosome loss, translocations or truncations. When DSBs occur, many proteins are recruited to the break site; these proteins serve to both initiate DNA repair and to activate a checkpoint response. Repair occurs via one of two pathways: non-homologous end-joining (NHEJ), in which broken DNA ends are directly ligated; or homologous recombination (HR), in which a homologous chromosome is used as a template in a replicative repair process. The checkpoint response is mediated by the phosphatidyl inositol 3-kinase-like kinases, Mec1 and Tel1 (ATR and ATM in humans, respectively). Two recent studies in yeast have significantly increased our understanding of when each of the proteins involved in these processes is localized to a break and, in addition, how their sequential localization is achieved. Specifically, these studies support and expand upon a model in which Tel1 and the NHEJ proteins are the first proteins to localize to the break to initiate signaling and attempt repair, but are subsequently replaced by Mec1 and the HR proteins. This transition is mediated by a cyclin-dependent kinase-dependent initiation of 5'-->3' processing (resection) of the DSB. Thus, the cell-cycle stage at which DSBs occur affects the way in which the DSBs are processed and recognized.

Animals↗

PubMed2004

DNA breaks promote genomic instability by impeding proper chromosome segregation.

BACKGROUND: Unrepaired DNA double-stranded breaks (DSBs) can result in the whole or partial loss of chromosomes. Previously, we showed that the ends of broken chromosomes remain associated. Here, we have examined the machinery that holds broken chromosome ends together, and we have explored the behavior of broken chromosomes as they pass through mitosis. RESULTS: Using GFP-localized arrays flanking an HO endonuclease site, we examined the association of broken chromosome ends in yeast cells that are checkpoint-arrested in metaphase. This association is partially dependent upon Rad50 and Rad52. After 6-8 hr, cells adapted to the checkpoint and resumed mitosis, segregating the broken chromosome. When this occurred, we found that the acentric fragments cosegregated into either the mother or daughter cell 95% of the time. Similarly, pedigree analysis showed that postmitotic repair of a broken chromosome (rejoining the centric and acentric fragments) occurred in either the mother or daughter cell, but rarely both, consistent with a model in which both acentric sister chromatid fragments are passaged into the same nucleus. CONCLUSIONS: These data suggest two related phenomena: an intrachromosomal association that holds the halves of a single broken sister chromatid together in metaphase and an interchromosomal force that tethers broken sister chromatids to each other and promotes their missegregation. Strikingly, the interchromosomal association of DNA breaks also promotes the missegregation of centromeric chromosomal fragments, albeit to a lesser extent than acentric fragments. The DNA break-induced missegregation of acentric and centric chromosome fragments provides a novel mechanism for the loss of heterozygosity that precedes tumorigenesis in mammalian cells.

Chromosome Breakage↗

PubMed2004

Cycling without the cyclosome: modeling a yeast strain lacking the APC.

The construction of viable Saccharomyces cerevisiae strains that lack the anaphase promoting complex (APC) was recently reported. The normally lethal deletions of APC genes were suppressed by the double deletion of the PDS1 and CLB5 genes in conjunction with the insertion of multiple copies of the SIC1 gene controlled by its endogenous promoter. It was proposed that cyclic expression and degradation of Sic1 results in oscillations of Clb/CDK activity necessary for the cell cycle. We have used an updated version of a mathematical model of the yeast cell cycle to model strains that lack the APC. With a few modifications, the model accurately simulates the viability of Apc- strains, as well as the phenotypes of 27 other previously characterized strains. We discuss a few minor inconsistencies between the model and experiment, and how these may inform future revisions to the model.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2003

Securin and B-cyclin/CDK are the only essential targets of the APC.

The anaphase-promoting complex/cyclosome (APC) is a highly conserved ubiquitin ligase that controls passage through the cell cycle by targeting many proteins for proteolysis. The complex is composed of at least thirteen core subunits, eight of which are essential, and two activating subunits, Cdc20 (essential) and Cdh1/Hct1 (non-essential). Previously, it was not known which APC targets are sufficient to explain the essential nature of the complex. Here, we show that each of the eight normally essential APC subunits is rendered non-essential ('bypass-suppressed') by the simultaneous removal/inhibition of the APC substrates securin (Pds1) and B-type cyclin/CDK (Clb/CDK). In strains lacking the APC, levels of Clb2 and Clb3 remain constant, but Clb/CDK activity oscillates as cells cycle. This suggests that in the absence of B-type cyclin destruction, oscillation of the Clb/CDK-inhibitor Sic1 is sufficient to trigger the feedback loops necessary for the bi-stable nature of Clb/CDK activity. These results strongly suggest that securin and B-type cyclin/CDK activity are the only obligatory targets of the APC in Saccharomyces cerevisiae.

Anaphase-Promoting Complex-Cyclosome↗