Cyclin-dependent kinase 1-dependent activation of APC/C ubiquitin ligase.

Error-free genome duplication and segregation are ensured through the timely activation of ubiquitylation enzymes. The anaphase-promoting complex or cyclosome (APC/C), a multisubunit E3 ubiquitin ligase, is regulated by phosphorylation. However, the mechanism remains elusive. Using systematic reconstitution and analysis of vertebrate APC/Cs under physiological conditions, we show how cyclin-dependent kinase 1 (CDK1) activates the APC/C through coordinated phosphorylation between Apc3 and Apc1. Phosphorylation of the loop domains by CDK1 in complex with p9/Cks2 (a CDK regulatory subunit) controlled loading of coactivator Cdc20 onto APC/C. A phosphomimetic mutation introduced into Apc1 allowed Cdc20 to increase APC/C activity in interphase. These results define a previously unrecognized subunit-subunit communication over a distance and the functional consequences of CDK phosphorylation. Cdc20 is a potential therapeutic target, and our findings may facilitate the development of specific inhibitors.

The transcription factor Atf1 binds and activates the APC/C ubiquitin ligase in fission yeast.

Fission yeast Atf1 is a member of the ATF/CREB basic leucine zipper (bZIP) family of transcription factors with strong homology to mammalian ATF2. Atf1 regulates transcription in response to stress stimuli and also plays a role in controlling heterochromatin formation and recombination. However, its DNA binding independent role is poorly studied. Here, we report that Atf1 has a distinct role in regulating the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. We have identified atf1(+) as a dose-dependent suppressor of apc5-1, a mutation causing mitotic arrest. Remarkably, the suppression is not dependent upon the bZIP domain and is therefore independent of the ability of Atf1 to bind DNA. Interestingly, Atf1 physically binds the APC/C in vivo. Furthermore, we show that addition of purified Atf1 proteins into a cell-free system stimulates ubiquitylation of cyclin B and securin by the APC/C. These results reveal a novel role for Atf1 in cell cycle control through protein-protein interaction.

In vitro assays for the anaphase-promoting complex/cyclosome (APC/C) in Xenopus egg extracts.

The anaphase-promoting complex/cyclosome (APC/C), a large (20S) multisubunit E3 ligase, has an essential role to ubiquitylate numerous substrates at specific times during mitosis and G1 phase as well as in meiosis. The deregulation of the APC/C causes cell death or genomic instability, which is a hallmark of cancers. Although 13 years have passed since its discovery, the molecular mechanisms of the APC/C-dependent ubiquitylation and proteolysis are still poorly understood. The development of in vitro systems enables the identification of new substrates and investigation of the molecular mechanisms by which the APC/C recognizes its substrates. This chapter describes in vitro assays reconstituted in Xenopus egg extracts.

The anaphase-promoting complex/cyclosome controls repair and recombination by ubiquitylating Rhp54 in fission yeast.

Homologous recombination (HR) is important for maintaining genome integrity and for the process of meiotic chromosome segregation and the generation of variation. HR is regulated throughout the cell cycle, being prevalent in the S and G2 phases and suppressed in the G1 phase. Here we show that the anaphase-promoting complex/cyclosome (APC/C) regulates homologous recombination in the fission yeast Schizosaccharomyces pombe by ubiquitylating Rhp54 (an ortholog of Rad54). We show that Rhp54 is a novel APC/C substrate that is destroyed in G1 phase in a KEN-box- and Ste9/Fizzy-related manner. The biological consequences of failing to temporally regulate HR via Rhp54 degradation are seen in haploid cells only in the absence of antirecombinase Srs2 function and are more extensive in diploid cells, which become sensitive to a range of DNA-damaging agents, including hydroxyurea, methyl methanesulfonate, bleomycin, and UV. During meiosis, expression of nondegradable Rhp54 inhibits interhomolog recombination and stimulates sister chromatid recombination. We thus propose that it is critical to control levels of Rhp54 in G1 to suppress HR repair of double-strand breaks and during meiosis to coordinate interhomolog recombination.

The Williams syndrome transcription factor interacts with PCNA to target chromatin remodelling by ISWI to replication foci.

Chromatin states have to be faithfully duplicated during DNA replication to maintain cell identity. It is unclear whether or how ATP-dependent chromatin-remodelling factors are involved in this process. Here we provide evidence that the Williams syndrome transcription factor (WSTF) is targeted to replication foci through direct interaction with the DNA clamp PCNA, an important coordinator of DNA and chromatin replication. WSTF, in turn, recruits imitation switch (ISWI)-type nucleosome-remodelling factor SNF2H to replication sites. These findings reveal a novel recruitment mechanism for ATP-dependent chromatin-remodelling factors that is fundamentally different from the previously documented targeting by sequence-specific transcriptional regulators. RNA-interference-mediated depletion of WSTF or SNF2H causes a compaction of newly replicated chromatin and increases the amount of heterochromatin markers, including HP1beta. This increase in the amount of HP1beta protein is mediated by progression through S phase and is not the result of an increase in HP1beta mRNA levels. We propose that the WSTF-ISWI complex has a role in the maintenance of chromatin structures during DNA replication.

The histone-fold protein complex CHRAC-15/17 enhances nucleosome sliding and assembly mediated by ACF.

The histone fold is a structural motif with which two related proteins interact and is found in complexes involved in wrapping DNA, the nucleosome, and transcriptional regulation, as in NC2. We reveal a novel function for histone-fold proteins: facilitation of nucleosome remodeling. ACF1-ISWI complex (ATP-dependent chromatin assembly and remodeling factor [ACF]) associates with histone-fold proteins (CHRAC-15 and CHRAC-17 in the human chromatin accessibility complex [CHRAC]) whose functional relevance has been unclear. We show that these histone-fold proteins facilitate ATP-dependent nucleosome sliding by ACF. Direct interaction of the CHRAC-15/17 complex with the ACF1 subunit is essential for this process. CHRAC-17 interacts with another histone-fold protein, p12, in DNA polymerase epsilon, but CHRAC-15 is essential for interaction with ACF and enhancement of nucleosome sliding. Surprisingly, CHRAC-15/17, p12/CHRAC-17, and NC2 complexes facilitate ACF-mediated chromatin assembly by a mechanism different from nucleosome sliding enhancement, suggesting a general activity of H2A/H2B type histone-fold complexes in chromatin assembly.