SUMOylation of the C-terminal domain of DNA topoisomerase IIα regulates the centromeric localization of Claspin

DNA topoisomerase II (TopoII) regulates DNA topology by its strand passaging reaction, which is required for genome maintenance by resolving tangled genomic DNA. In addition, TopoII contributes to the structural integrity of mitotic chromosomes and to the activation of cell cycle checkpoints in mitosis. Post-translational modification of TopoII is one of the key mechanisms by which its broad functions are regulated during mitosis. SUMOylation of TopoII is conserved in eukaryotes and plays a critical role in chromosome segregation. Using Xenopus laevis egg extract, we demonstrated previously that TopoIIα is modified by SUMO on mitotic chromosomes and that its activity is modulated via SUMOylation of its lysine at 660. However, both biochemical and genetic analyses indicated that TopoII has multiple SUMOylation sites in addition to Lys660, and the functions of the other SUMOylation sites were not clearly determined. In this study, we identified the SUMOylation sites on the C-terminal domain (CTD) of TopoIIα. CTD SUMOylation did not affect TopoIIα activity, indicating that its function is distinct from that of Lys660 SUMOylation. We found that CTD SUMOylation promotes protein binding and that Claspin, a well-established cell cycle checkpoint mediator, is one of the SUMOylation-dependent binding proteins. Claspin harbors 2 SUMO-interacting motifs (SIMs), and its robust association to mitotic chromosomes requires both the SIMs and TopoIIα-CTD SUMOylation. Claspin localizes to the mitotic centromeres depending on mitotic SUMOylation, suggesting that TopoIIα-CTD SUMOylation regulates the centromeric localization of Claspin. Our findings provide a novel mechanistic insight regarding how TopoIIα-CTD SUMOylation contributes to mitotic centromere activity.     

SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis

Polo-like kinase 1 (Plk1)-interacting checkpoint helicase (PICH) localizes at the centromere and is critical for proper chromosome segregation during mitosis. However, the precise molecular mechanism of PICH's centromeric localization and function at the centromere is not yet fully understood. Recently, using Xenopus egg extract assays, we showed that PICH is a promiscuous SUMO binding protein. To further determine the molecular consequence of PICH/SUMO interaction on PICH function, we identified 3 SUMO-interacting motifs (SIMs) on PICH and generated a SIM-deficient PICH mutant. Using the conditional expression of PICH in cells, we found distinct roles of PICH SIMs during mitosis. Although all SIMs are dispensable for PICH's localization on ultrafine anaphase DNA bridges, only SIM3 (third SIM, close to the C-terminus end of PICH) is critical for its centromeric localization. Intriguingly, the other 2 SIMs function in chromatin bridge prevention. With these results, we propose a novel SUMO-dependent regulation of PICH's function on mitotic centromeres.     

On the regulation,function, and localization of the DNA-dependent ATPase PICH

The putative chromatin remodeling enzyme Plk1-interacting checkpoint helicase (PICH) was discovered as an interaction partner and substrate of the mitotic kinase Plk1. During mitosis PICH associates with centromeres and kinetochores and, most interestingly, constitutes a robust marker for ultrafine DNA bridges (UFBs) that connect separating chromatids in anaphase cells. The precise roles of PICH remain to be clarified. Here, we have used antibody microinjection and siRNA-rescue experiments to study PICH function and localization during M phase progression, with particular emphasis on the role of the predicted ATPase domain and the regulation of PICH localization by Plk1. We show that interference with PICH function results in chromatin bridge formation and micronucleation and that ATPase activity is critical for PICH function. Interestingly, an intact ATPase domain of PICH is required for prevention of chromatin bridge formation but not for UFB resolution, and quantitative analyses of UFB and chromatin bridge frequencies suggest that these structures are of different etiologies. We also show that the ATPase activity of PICH is required for temporal and spatial control of PICH localization to chromatin and that Plk1 likely controls PICH localization through phosphorylation of proteins distinct from PICH itself. This work strengthens the view that PICH is an important, Plk1-regulated enzyme, whose ATPase activity is essential for maintenance of genome integrity. Although not required for the spindle assembly checkpoint, PICH is clearly important for faithful chromosome segregation.     

Biochemical heterogeneity and developmental varieties in T-cell leukemia

During mitosis, chromosomes undergo dynamic structural changes that include condensation of chromosomes – the formation of individual compact chromosomes necessary for faithful segregation of sister chromatids in anaphase. Polo-like kinase 1 (Plk1) regulates multiple mitotic events by binding to targeting factors at different mitotic structures in a phosphorylation dependent manner. In this study, we report the identification of a putative ATPase that targets Plk1 to chromosome arms during mitosis. PICH (Plk1-interacting checkpoint “helicase”) displays a temporal localization on chromosome arms and kinetochores during early mitosis. Interaction with PICH recruits Plk1 to chromosome arms and disruption of this interaction abolishes Plk1 localization on chromosome arms. Moreover, depletion of PICH or overexpression of PICH mutant that is defective in Plk1 binding or ATP binding causes defects in mitotic chromosome compaction, formation of anaphase bridge and cytokinesis failure. We provide data to show that both PICH phosphorylation and its ATPase activity are required for mitotic chromosome compaction. Our study provides a mechanism for targeting Plk1 to chromosome arms and suggests that the PICH ATPase activity is important for the regulation of mitotic chromosome architecture.     

End‐joining inhibition at telomeres requires the translocase and polySUMO‐dependent ubiquitin ligase Uls1

In eukaryotes, permanent inhibition of the non‐homologous end joining (NHEJ) repair pathway at telomeres ensures that chromosome ends do not fuse. In budding yeast, binding of Rap1 to telomere repeats establishes NHEJ inhibition. Here, we show that the Uls1 protein is required for the maintenance of NHEJ inhibition at telomeres. Uls1 protein is a non‐essential Swi2/Snf2‐related translocase and a Small Ubiquitin‐related Modifier (SUMO)‐Targeted Ubiquitin Ligase (STUbL) with unknown targets. Loss of Uls1 results in telomere–telomere fusions. Uls1 requirement is alleviated by the absence of poly‐SUMO chains and by rap1 alleles lacking SUMOylation sites. Furthermore, Uls1 limits the accumulation of Rap1 poly‐SUMO conjugates. We propose that one of Uls1 functions is to clear non‐functional poly‐SUMOylated Rap1 molecules from telomeres to ensure the continuous efficiency of NHEJ inhibition. Since Uls1 is the only known STUbL with a translocase activity, it can be the general molecular sweeper for the clearance of poly‐SUMOylated proteins on DNA in eukaryotes.     

A Role for SUMO in meiotic chromosome synapsis

During meiotic prophase, homologous chromosomes engage in a complex series of interactions that ensure their proper segregation at meiosis I. A central player in these interactions is the synaptonemal complex (SC), a proteinaceous structure elaborated along the lengths of paired homologs. In mutants that fail to make SC, crossing over is decreased, and chromosomes frequently fail to recombine; consequently, many meiotic products are inviable because of aneuploidy. Here, we have investigated the role of the small ubiquitin-like protein modifier (SUMO) in SC formation during meiosis in budding yeast. We show that SUMO localizes specifically to synapsed regions of meiotic chromosomes and that this localization depends on Zip1, a major building block of the SC. A non-null allele of the UBC9 gene, which encodes the SUMO-conjugating enzyme, impairs Zip1 polymerization along chromosomes. The Ubc9 protein localizes to meiotic chromosomes, coincident with SUMO staining. In the zip1 mutant, SUMO localizes to discrete foci on chromosomes. These foci coincide with axial associations, where proteins involved in synapsis initiation are located. Our data suggest a model in which SUMO modification of chromosomal proteins promotes polymerization of Zip1 along chromosomes. The ubc9 mutant phenotype provides the first evidence for a cause-and-effect relationship between sumoylation and synapsis.     

Rev7/Mad2B plays a critical role in the assembly of a functional mitotic spindle

The spindle assembly checkpoint (SAC) acts as a guardian against cellular threats that may lead to chromosomal missegregation and aneuploidy. Mad2, an anaphase-promoting complex/cyclosome-Cdc20 (APC/C^Cdc20) inhibitor, has an additional homolog in mammals known as Mad2B, Mad2L2 or Rev7. Apart from its role in Polζ-mediated translesion DNA synthesis and double-strand break repair, Rev7 is also believed to inhibit APC/C by negatively regulating Cdh1. Here we report yet another function of Rev7 in cultured human cells. Rev7, as predicted earlier, is involved in the formation of a functional spindle and maintenance of chromosome segregation. In the absence of Rev7, cells tend to arrest in G2/M-phase and display increased monoastral and abnormal spindles with misaligned chromosomes. Furthermore, Rev7-depleted cells show Mad2 localization at the kinetochores of metaphase cells, an indicator of activated SAC, coupled with increased levels of Cyclin B1, an APC^Cdc20 substrate. Surprisingly unlike Mad2, depletion of Rev7 in several cultured human cell lines did not compromise SAC activity. Our data therefore suggest that besides its role in APC/C^Cdh1 inhibition, Rev7 is also required for mitotic spindle organization and faithful chromosome segregation most probably through its physical interaction with RAN.     

Proteomic analysis of human metaphase chromosomes reveals topoisomerase II alpha as an Aurora B substrate

The essential Aurora B kinase is a chromosomal passenger protein that is required for mitotic chromosome alignment and segregation. Aurora B function is dependent on the chromosome passenger, INCENP. INCENP, in turn, requires sister chromatid cohesion for its appropriate behaviour. Relatively few substrates have been identified for Aurora B, so that the precise role it plays in controlling mitosis remains to be elucidated. To identify potential novel mitotic substrates of Aurora B, extracted chromosomes were prepared from mitotically-arrested HeLa S3 cells and incubated with recombinant human Aurora B in the presence of radioactive ATP. Immunoblot analysis confirmed the HeLa scaffold fraction to be enriched for known chromosomal proteins including CENP-A, CENP-B, CENP-C, ScII and INCENP. Mass spectrometry of bands excised from one-dimensional polyacrylamide gels further defined the protein composition of the extracted chromosome fraction. Cloning, fluorescent tagging and expression in HeLa cells of the putative GTP-binding protein NGB/CRFG demonstrated it to be a novel mitotic chromosome protein, with a perichromosomal localisation. Identi fication of the protein bands corresponding to those phosphorylated by Aurora B revealed topoisomerase II alpha (topo IIα) as a potential Aurora B substrate. Purified recombinant human topo IIα was phosphorylated by Aurora B in vitro, confirming this proteomic approach as a valid method for the initial definition of candidate substrates of key mitotic kinases.     

Subcellular Localization of Bacillus subtilis SMC, a Protein Involved in Chromosome Condensation and Segregation

We have investigated the subcellular localization of the SMC protein in the gram-positive bacterium Bacillus subtilis. Recent work has shown that SMC is required for chromosome condensation and faithful chromosome segregation during the B. subtilis cell cycle. Using antibodies against SMC and fluorescence microscopy, we have shown that SMC is associated with the chromosome but is also present in discrete foci near the poles of the cell. DNase treatment of permeabilized cells disrupted the association of SMC with the chromosome but not with the polar foci. The use of a truncated smc gene demonstrated that the C-terminal domain of the protein is required for chromosomal binding but not for the formation of polar foci. Regular arrays of SMC-containing foci were still present between nucleoids along the length of aseptate filaments generated by depleting cells of the cell division protein FtsZ, indicating that the formation of polar foci does not require the formation of septal structures. In slowly growing cells, which have only one or two chromosomes, SMC foci were principally observed early in the cell cycle, prior to or coincident with chromosome segregation. Cell cycle-dependent release of stored SMC from polar foci may mediate segregation by condensation of chromosomes.     

Smc5/6-mediated regulation of replication progression contributes to chromosome assembly during mitosis in human cells

The structural maintenance of chromosomes (SMC) proteins constitute the core of critical complexes involved in structural organization of chromosomes. In yeast, the Smc5/6 complex is known to mediate repair of DNA breaks and replication of repetitive genomic regions, including ribosomal DNA loci and telomeres. In mammalian cells, which have diverse genome structure and scale from yeast, the Smc5/6 complex has also been implicated in DNA damage response, but its further function in unchallenged conditions remains elusive. In this study, we addressed the behavior and function of Smc5/6 during the cell cycle. Chromatin fractionation, immunofluorescence, and live-cell imaging analyses indicated that Smc5/6 associates with chromatin during interphase but largely dissociates from chromosomes when they condense in mitosis. Depletion of Smc5 and Smc6 resulted in aberrant mitotic chromosome phenotypes that were accompanied by the abnormal distribution of topoisomerase IIα (topo IIα) and condensins and by chromosome segregation errors. Importantly, interphase chromatin structure indicated by the premature chromosome condensation assay suggested that Smc5/6 is required for the on-time progression of DNA replication and subsequent binding of topo IIα on replicated chromatids. These results indicate an essential role of the Smc5/6 complex in processing DNA replication, which becomes indispensable for proper sister chromatid assembly in mitosis.     

BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges

Mutations in BLM cause Bloom's syndrome, a disorder associated with cancer predisposition and chromosomal instability. We investigated whether BLM plays a role in ensuring the faithful chromosome segregation in human cells. We show that BLM-defective cells display a higher frequency of anaphase bridges and lagging chromatin than do isogenic corrected derivatives that eptopically express the BLM protein. In normal cells undergoing mitosis, BLM protein localizes to anaphase bridges, where it colocalizes with its cellular partners, topoisomerase IIIalpha and hRMI1 (BLAP75). Using BLM staining as a marker, we have identified a class of ultrafine DNA bridges in anaphase that are surprisingly prevalent in the anaphase population of normal human cells. These so-called BLM-DNA bridges, which also stain for the PICH protein, frequently link centromeric loci, and are present at an elevated frequency in cells lacking BLM. On the basis of these results, we propose that sister-chromatid disjunction is often incomplete in human cells even after the onset of anaphase. We present a model for the action of BLM in ensuring complete sister chromatid decatenation in anaphase.     

Topoisomerase II Regulates the Maintenance of DNA Methylation

The maintenance of DNA methylation in nascent DNA is a critical event for numerous biological processes. Following DNA replication, DNMT1 is the key enzyme that strictly copies the methylation pattern from the parental strand to the nascent DNA. However, the mechanism underlying this highly specific event is not thoroughly understood. In this study, we identified topoisomerase IIα (TopoIIα) as a novel regulator of the maintenance DNA methylation. UHRF1, a protein important for global DNA methylation, interacts with TopoIIα and regulates its localization to hemimethylated DNA. TopoIIα decatenates the hemimethylated DNA following replication, which might facilitate the methylation of the nascent strand by DNMT1. Inhibiting this activity impairs DNA methylation at multiple genomic loci. We have uncovered a novel mechanism during the maintenance of DNA methylation.     

Topoisomerase 3α and RMI1 Suppress Somatic Crossovers and Are Essential for Resolution of Meiotic Recombination Intermediates in Arabidopsis thaliana

Topoisomerases are enzymes with crucial functions in DNA metabolism. They are ubiquitously present in prokaryotes and eukaryotes and modify the steady-state level of DNA supercoiling. Biochemical analyses indicate that Topoisomerase 3α (TOP3α) functions together with a RecQ DNA helicase and a third partner, RMI1/BLAP75, in the resolution step of homologous recombination in a process called Holliday Junction dissolution in eukaryotes. Apart from that, little is known about the role of TOP3α in higher eukaryotes, as knockout mutants show early lethality or strong developmental defects. Using a hypomorphic insertion mutant of Arabidopsis thaliana (top3α-2), which is viable but completely sterile, we were able to define three different functions of the protein in mitosis and meiosis. The top3α-2 line exhibits fragmented chromosomes during mitosis and sensitivity to camptothecin, suggesting an important role in chromosome segregation partly overlapping with that of type IB topoisomerases. Furthermore, AtTOP3α, together with AtRECQ4A and AtRMI1, is involved in the suppression of crossover recombination in somatic cells as well as DNA repair in both mammals and A. thaliana. Surprisingly, AtTOP3α is also essential for meiosis. The phenotype of chromosome fragmentation, bridges, and telophase I arrest can be suppressed by AtSPO11 and AtRAD51 mutations, indicating that the protein is required for the resolution of recombination intermediates. As Atrmi1 mutants have a similar meiotic phenotype to Attop3α mutants, both proteins seem to be involved in a mechanism safeguarding the entangling of homologous chromosomes during meiosis. The requirement of AtTOP3α and AtRMI1 in a late step of meiotic recombination strongly hints at the possibility that the dissolution of double Holliday Junctions via a hemicatenane intermediate is indeed an indispensable step of meiotic recombination.     

Drosophila Casein Kinase I Alpha Regulates Homolog Pairing and Genome Organization by Modulating Condensin II Subunit Cap-H2 Levels

The spatial organization of chromosomes within interphase nuclei is important for gene expression and epigenetic inheritance. Although the extent of physical interaction between chromosomes and their degree of compaction varies during development and between different cell-types, it is unclear how regulation of chromosome interactions and compaction relate to spatial organization of genomes. Drosophila is an excellent model system for studying chromosomal interactions including homolog pairing. Recent work has shown that condensin II governs both interphase chromosome compaction and homolog pairing and condensin II activity is controlled by the turnover of its regulatory subunit Cap-H2. Specifically, Cap-H2 is a target of the SCF^Slimb E3 ubiquitin-ligase which down-regulates Cap-H2 in order to maintain homologous chromosome pairing, chromosome length and proper nuclear organization. Here, we identify Casein Kinase I alpha (CK1α) as an additional negative-regulator of Cap-H2. CK1α-depletion stabilizes Cap-H2 protein and results in an accumulation of Cap-H2 on chromosomes. Similar to Slimb mutation, CK1α depletion in cultured cells, larval salivary gland, and nurse cells results in several condensin II-dependent phenotypes including dispersal of centromeres, interphase chromosome compaction, and chromosome unpairing. Moreover, CK1α loss-of-function mutations dominantly suppress condensin II mutant phenotypes in vivo. Thus, CK1α facilitates Cap-H2 destruction and modulates nuclear organization by attenuating chromatin localized Cap-H2 protein.     

PICH and Cotargeted Plk1 Coordinately Maintain Prometaphase Chromosome Arm Architecture

To maintain genomic stability, chromosome architecture needs to be tightly regulated as chromosomes undergo condensation during prophase and separation during anaphase, but the mechanisms remain poorly understood. Here, we show that the Plk1-binding protein PICH and Plk1 kinase coordinately maintain chromosome architecture during prometaphase. PICH knockdown results in a loss of Plk1 from the chromosome arm and an increase in highly disorganized “wavy” chromosomes that exhibit an “open” or “X-shaped” configuration, consistent with a loss of chromosome arm cohesion. Such chromosome disorganization occurs with essentially no change in the localization of condensin or cohesin on chromosomes. Interestingly, the chromosome disorganization could be prevented by treatment with a topoisomerase II inhibitor ICRF-193, suggesting that the PICH–Plk1 complex normally maintains chromosome architecture in a manner that involves topoisomerase II activity. PICH knockdown does not affect initial chromosome compaction at prophase but causes anaphase DNA bridge formation and failed abscission. Our studies suggest that the PICH–Plk1 complex plays a critical role in maintaining prometaphase chromosome architecture.