SummaryBubR1 is a central component of the spindle assembly checkpoint that inhibits progression into anaphase in response to improper kinetochore-microtubule interactions. In addition, BubR1 also helps stabilize kinetochore-microtubule interactions by counteracting the Aurora B kinase but the mechanism behind this is not clear. Here we show that BubR1 directly binds to the B56 family of protein phosphatase 2A (PP2A) regulatory subunits through a conserved motif that is phosphorylated by cyclin-dependent kinase 1 (Cdk1) and polo-like kinase 1 (Plk1). Two highly conserved hydrophobic residues surrounding the serine 670 Cdk1 phosphorylation site are required for B56 binding. Mutation of these residues prevents the establishment of a proper metaphase plate and delays cells in mitosis. Furthermore, we show that phosphorylation of serines 670 and 676 stimulates the binding of B56 to BubR1 and that BubR1 targets a pool of B56 to kinetochores. Our data suggest that BubR1 counteracts Aurora B kinase activity at improperly attached kinetochores by recruiting B56-PP2A phosphatase complexes.

Activation of oncogenes or inhibition of WEE1 kinase deregulates cyclin-dependent kinase (CDK) activity and leads to replication stress; however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibition of WEE1 kinase rapidly increases initiation of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted. Furthermore, addition of nucleosides counteracts the effects of unscheduled CDK activity on fork speed and DNA DSB formation. Finally, we show that WEE1 regulates the ionizing radiation (IR)-induced S-phase checkpoint, consistent with its role in control of replication initiation. In conclusion, these results suggest that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage. DNA replication is tightly monitored to ensure that the genome is replicated precisely once per cell cycle and that DNA replication is complete before mitosis begins. Conditions for DNA synthesis are rarely ideal, and a number of obstacles must often be dealt with, such as a damaged DNA template and shortage of deoxynucleoside triphosphates (dNTPs), to allow replication fork progression. Stalling replication forks pose serious threats to genome integrity because they can collapse through disassembly of the replication complex and break (6,11,24). Such damaged forks may subsequently undergo incorrect repair, leading to genetic changes like chromosomal rearrangements (6,24). Recent data have also revealed that activated oncogenes can induce DNA replication stress, defined here as replication-associated DNA damage (2, 3, 10). Oncogene-induced replication stress can lead to additional tumor-promoting genetic changes, but it may also serve as a tumor barrier by activation of cell cycle arrest, apoptosis, and/or senescence during early tumor development (32).WEE1 and CHK1 kinases have major roles in suppressing DNA replication stress (4,23,27,42), and attenuation of their function can contribute to carcinogenesis and cause cell death (40). The massive amount of DNA breakage is likely mediated by DNA endonuclease activity, and recent studies suggest that this is mediated by the endonuclease MUS81 (12,14,15). Notably, the mechanisms by which oncogenes or inhibition of checkpoint kinases can lead to endonuclease-mediated DNA breakage are poorly understood. It is also not fully understood if these breaks also play a role in inducing fork stalling or if they are temporally delayed events secondary to the fork stalling.As both oncogenes and checkpoint kinases are regulators of cyclin-dependent kinase (CDK) activity, we previously proposed that most of the DNA replication str...

WEE1 and CHK1 jointly regulate Cdk activity to prevent DNA damage during replication.

Degradation of the histone H4 methyltransferase SET8, which regulates chromosome compaction and genomic integrity, is regulated by the CRL4(CDT2) ubiquitin ligase to facilitate DNA replication and repair.

Highlights d The conserved PP4 holoenzyme binds to FxxP motifs that provide specificity d FxxP motifs bind to a conserved binding pocket on PP4 regulatory subunit d Binding to FxxP motifs can be regulated through phosphorylation d PP4 binding to an FxxP motif in WAPL regulates its cohesin release activity

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying entry into anaphase until all sister chromatids have become bi-oriented. A key component of the SAC is the Mad2 protein, which can adopt either an inactive open (O-Mad2) or active closed (C-Mad2) conformation. The conversion of O-Mad2 into C-Mad2 at unattached kinetochores is thought to be a key step in activating the SAC. The template model proposes that this is achieved by the recruitment of soluble O-Mad2 to C-Mad2 bound at kinetochores through its interaction with Mad1. Whether Mad1 has additional roles in the SAC beyond recruitment of C-Mad2 to kinetochores has not yet been addressed. Here, we show that Mad1 is required for mitotic arrest even when C-Mad2 is artificially recruited to kinetochores, indicating that it has indeed an additional function in promoting the checkpoint. The C-terminal globular domain of Mad1 and conserved residues in this region are required for this unexpected function of Mad1.

SummaryThe anaphase-promoting complex/cyclosome (APC/C) and the spindle assembly checkpoint (SAC), which inhibits the APC/C, are essential determinants of mitotic timing and faithful division of genetic material. Activation of the APC/C is known to depend on two APC/C-interacting E2 ubiquitin-conjugating enzymes—UBE2C and UBE2S. We show that APC/C activity in human cells is tuned by the combinatorial use of three E2s, namely UBE2C, UBE2S, and UBE2D. Genetic deletion of UBE2C and UBE2S, individually or in combination, leads to discriminative reduction in APC/C function and sensitizes cells to UBE2D depletion. Reduction of APC/C activity results in loss of switch-like metaphase-to-anaphase transition and, strikingly, renders cells insensitive to chemical inhibition of MPS1 and genetic ablation of MAD2, both of which are essential for the SAC. These results provide insights into the regulation of APC/C activity and demonstrate that the essentiality of the SAC is imposed by the strength of the APC/C.

BackgroundGlobal residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained via screening of reduced-size ensembles.Methodology/Principal FindingsThe strategy involves combining a sequential mutagenesis scheme to reduce library size with structurally stabilizing mutations, chaperone complementation, and reduced temperature of gene expression. In six steps, we eliminated a common buried residue, Phe, from the green fluorescent protein (GFP), while retaining activity. A GFP variant containing 11 Phe residues was used as starting scaffold to generate 10 separate variants in which each Phe was replaced individually (in one construct two adjacent Phe residues were changed simultaneously), while retaining varying levels of activity. Combination of these substitutions to generate a Phe-free variant of GFP abolished fluorescence. Combinatorial re-introduction of five Phe residues, based on the activities of the respective single amino acid replacements, was sufficient to restore GFP activity. Successive rounds of mutagenesis generated active GFP variants containing, three, two, and zero Phe residues. These GFPs all displayed progenitor-like fluorescence spectra, temperature-sensitive folding, a reduced structural stability and, for the least stable variants, a reduced steady state abundance.Conclusions/SignificanceThe results provide strategies for the design of novel GFP reporters. The described approach offers a means to enable engineering of active proteins that lack certain amino acids, a key step towards expanding the functional repertoire of uniquely labeled proteins in synthetic biology.