Jan-Michael Peters

Division Machinery Tagged: An international consortium of labs has been testing the feasibility of large-scale screening for insights into the function of mammalian proteins by expressing a tagged version of proteins from bacterial artificial chromosomes harbored in mammalian cells. Depending on the tag used, Hutchins et al. (p. 593 , published online 1 April) were able to monitor localization of tagged proteins by microscopy or to isolate interacting proteins and subsequently identify the binding partners by mass spectrometry. Applying the technology to proteins implicated in control of cell division revealed about 100 protein machines required for mitosis.

Cohesin complexes mediate sister chromatid cohesion. Cohesin also becomes enriched at DNA double-strand break sites and facilitates recombinational DNA repair. Here, we report that cohesin is essential for the DNA damage-induced G2/M checkpoint. In contrast to cohesin's role in DNA repair, the checkpoint function of cohesin is independent of its ability to mediate cohesion. After RNAi-mediated depletion of cohesin, cells fail to properly activate the checkpoint kinase Chk2 and have defects in recruiting the mediator protein 53BP1 to DNA damage sites. Earlier work has shown that phosphorylation of the cohesin subunits Smc1 and Smc3 is required for the intra-S checkpoint, but Smc1/Smc3 are also subunits of a distinct recombination complex, RC-1. It was, therefore, unknown whether Smc1/Smc3 function in the intra-S checkpoint as part of cohesin. We show that Smc1/Smc3 are phosphorylated as part of cohesin and that cohesin is required for the intra-S checkpoint. We propose that accumulation of cohesin at DNA break sites is not only needed to mediate DNA repair, but also facilitates the recruitment of checkpoint proteins, which activate the intra-S and G2/M checkpoints.

Once all chromosomes are connected to the mitotic spindle (bioriented), anaphase is initiated by the protein ubiquitylation activity of the anaphase-promoting complex/cyclosome (APC/C) and its coactivator Cdc20 (APC/C ^Cdc20 ). Before chromosome biorientation, anaphase is delayed by a mitotic checkpoint complex (MCC) that inhibits APC/C ^Cdc20 . We used single-particle electron microscopy to obtain three-dimensional models of human APC/C in various functional states: bound to MCC, to Cdc20, or to neither (apo-APC/C). These experiments revealed that MCC associates with the Cdc20 binding site on APC/C, locks the otherwise flexible APC/C in a “closed” state, and prevents binding and ubiquitylation of a wide range of different APC/C substrates. These observations clarify the structural basis for the inhibition of APC/C by spindle checkpoint proteins.

Sister chromatids are physically connected by cohesin complexes. This sister chromatid cohesion is essential for the biorientation of chromosomes on the mitotic and meiotic spindle. In many species, cohesion between chromosome arms is partly dissolved in prophase of mitosis, whereas cohesion is protected at centromeres until the onset of anaphase. In vertebrates, the protein Sgo1, protein phosphatase 2A, and several other proteins are required for protection of centromeric cohesin in early mitosis. In fission yeast, the recruitment of heterochromatin protein Swi6/HP1 to centromeres by the histone-methyltransferase Clr4/Suv39h is required for enrichment of cohesin at centromeres already in interphase. We have tested if the Suv39h–HP1 histone methylation pathway is also required for enrichment and mitotic protection of cohesin at centromeres in mammalian cells. We show that cohesin and HP1 proteins partially colocalize at mitotic centromeres but that cohesin localization is not detectably altered in mouse embryonic fibroblasts that lack Suv39h genes and in which HP1 proteins can, therefore, not be properly enriched in pericentric heterochromatin. Our data indicate that the Suv39h–HP1 pathway is not essential for enrichment and mitotic protection of cohesin at centromeres in mammalian cells.

Cells respond to DNA damage by strengthening chromosome cohesion across the entire genome independently of DNA replication.

The anaphase promoting complex/cyclosome (APC/C) is a 1.5-MDa ubiquitin ligase complex that initiates sister-chromatid separation and exit from mitosis by targeting cyclin B and securin for destruction by the 26S proteasome. APC/C activity is also indirectly required for DNA replication, because APC/C-mediated cyclin degradation leads to the inactivation of cyclin-dependent kinase-1 (Cdk1), which is a prerequisite for the assembly of pre-replication complexes. APC/C is activated by proteins of the Cdc20/Cdh1 family. The interaction between APC/C and its co-activators is tightly controlled by phosphorylation and is restricted to mitosis and G1 phase. In addition, APC/C activity can be restrained by a number of inhibitory proteins. Mad2 and BubR1 inhibit APC/C during spindle assembly and thereby prevent precocious initiation of anaphase and exit from mitosis. Members of the early mitotic inhibitor-1 (EMI1)/regulator of cyclin A-1 (RCA1) family inhibit APC/C from S phase until early mitosis and during meiosis in vertebrate eggs. Co-activator proteins activate APC/C by facilitating the recruitment of substrates. All known APC/C co-activators contain a propeller-shaped WD40 domain that interacts with a recognition element in APC/C substrates and is known as the destruction box (D-box). Co-activators are required but not sufficient for substrate recognition because the APC/C subunit Doc1 is also needed for this process. Several observations suggest that the D-box of substrates might interact with both co-activators and APC/C subunits to form a ternary complex in which substrate ubiquitylation occurs.

Separase is a protease whose liberation from its inhibitory chaperone Securin triggers sister chromatid disjunction at anaphase onset in yeast by cleaving cohesin's kleisin subunit. We have created conditional knockout alleles of the mouse Separase and Securin genes. Deletion of both copies of Separase but not Securin causes embryonic lethality. Loss of Securin reduces Separase activity because deletion of just one copy of the Separase gene is lethal to embryos lacking Securin. In embryonic fibroblasts, Separase depletion blocks sister chromatid separation but does not prevent other aspects of mitosis, cytokinesis, or chromosome replication. Thus, fibroblasts lacking Separase become highly polyploid. Hepatocytes stimulated to proliferate in vivo by hepatectomy also become unusually large and polyploid in the absence of Separase but are able to regenerate functional livers. Separase depletion in bone marrow causes aplasia and the presumed death of hematopoietic cells other than erythrocytes. Destruction of sister chromatid cohesion by Separase may be a universal feature of mitosis in eukaryotic cells.