Chromosome condensation by a human condensin complex in Xenopus egg extracts

13S condensin is a five-subunit protein complex that plays a central role in mitotic chromosome condensation. The condensin complex was originally identified and purified from Xenopus egg extracts and shown to have an ATP-dependent positive supercoiling activity in vitro. We report here the characterization of a human condensin complex purified from HeLa cell nuclear extracts. The human 13S complex has exactly the same composition as its Xenopus counterpart, being composed of two structural maintenance of chromosomes (human chromosome-associated polypeptide (hCAP)-C and hCAP-E) subunits and three non-structural maintenance of chromosomes (hCAP-D2/CNAP1, hCAP-G, and hCAP-H/BRRN) subunits. Human condensin purified from asynchronous HeLa cell cultures fails to reconfigure DNA structure in vitro. When phosphorylated by purified cdc2-cyclin B, however, it gains the ability to introduce positive supercoils into DNA in the presence of ATP and topoisomerase I. Strikingly, human condensin can induce chromosome condensation when added back into a Xenopus egg extract that has been immunodepleted of endogenous condensin. Thus, the structure and function of the condensin complex are highly conserved between Xenopus and humans, underscoring its fundamental importance in mitotic chromosome dynamics in eukaryotic cells.     

13S condensin actively reconfigures DNA by introducing global positive writhe: implications for chromosome condensation.

Xenopus 13S condensin converts interphase chromatin into mitotic-like chromosomes, and, in the presence of ATP and a type I topoisomerase, introduces (+) supercoils into DNA. The specific production of (+) trefoil knots in the presence of condensin and a type II topoisomerase shows that condensin reconfigures DNA by introducing an ordered, global, (+) writhe. Knotting required ATP hydrolysis and cell cycle-specific phosphorylation of condensin. Condensin bound preferentially to (+) supercoiled DNA in the presence of ATP but not in its absence. Our results suggest a mechanism for the compaction of chromatin by condensin during mitosis.     

Negative regulation of condensin I by CK2-mediated phosphorylation

Condensin I, which plays an essential role in mitotic chromosome assembly and segregation in vivo, constrains positive supercoils into DNA in the presence of adenosine triphosphate in vitro. Condensin I is constitutively present in a phosphorylated form throughout the HeLa cell cycle, but the sites at which it is phosphorylated in interphase cells differ from those recognized by Cdc2 during mitosis. Immunodepletion, in vitro phosphorylation, and immunoblot analysis using a phospho-specific antibody suggested that the CK2 kinase is likely to be responsible for phosphorylation of condensin I during interphase. In contrast to the slight stimulatory effect of Cdc2-induced phosphorylation of condensin I on supercoiling, phosphorylation by CK2 reduced the supercoiling activity of condensin I. CK2-mediated phosphorylation of condensin I is spatially and temporally regulated in a manner different to that of Cdc2-mediated phosphorylation: CK2-dependent phosphorylation increases during interphase and decreases on chromosomes during mitosis. These findings are the first to demonstrate a negative regulatory mode for condensin I, a process that may influence chromatin structure during interphase and mitosis.     

Condensin aids sister chromatid decatenation by topoisomerase II

The condensin complex is a key determinant of mitotic chromosome architecture. In addition, condensin promotes resolution of sister chromatids during anaphase, a function that is conserved from prokaryotes to human. Anaphase bridges observed in cells lacking condensin are reminiscent of chromosome segregation failure after inactivation of topoisomerase II (topo II), the enzyme that removes catenanes persisting between sister chromatids following DNA replication. Circumstantial evidence has linked condensin to sister chromatid decatenation but, because of the difficulty of observing chromosome catenation, this link has remained indirect. Alternative models for how condensin facilitates chromosome resolution have been put forward. Here, we follow the catenation status of circular minichromosomes of three sizes during the Saccharomyeces cerevisiae cell cycle. Catenanes are produced during DNA replication and are for the most part swiftly resolved during and following S-phase, aided by sister chromatid separation. Complete resolution, however, requires the condensin complex, a dependency that becomes more pronounced with increasing chromosome size. Our results provide evidence that condensin prevents deleterious anaphase bridges during chromosome segregation by promoting sister chromatid decatenation.     

DNA supercoiling during ATP-dependent DNA translocation by the type I restriction enzyme EcoAI

Type I restriction enzymes cleave DNA at non-specific sites far from their recognition sequence as a consequence of ATP-dependent DNA translocation past the enzyme. During this reaction, the enzyme remains bound to the recognition sequence and translocates DNA towards itself simultaneously from both directions, generating DNA loops, which appear to be supercoiled when visualised by electron microscopy. To further investigate the mechanism of DNA translocation by type I restriction enzymes, we have probed the reaction intermediates with DNA topoisomerases. A DNA cleavage-deficient mutant of EcoAI, which has normal DNA translocation and ATPase activities, was used in these DNA supercoiling assays. In the presence of eubacterial DNA topoisomerase I, which specifically removes negative supercoils, the EcoAI mutant introduced positive supercoils into relaxed plasmid DNA substrate in a reaction dependent on ATP hydrolysis. The same DNA supercoiling activity followed by DNA cleavage was observed with the wild-type EcoAI endonuclease. Positive supercoils were not seen when eubacterial DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I, which removes both positive and negative supercoils. Furthermore, addition of eukaryotic DNA topoisomerase I to the product of the supercoiling reaction resulted in its rapid relaxation. These results are consistent with a model in which EcoAI translocation along the helical path of closed circular DNA duplex simultaneously generates positive supercoils ahead and negative supercoils behind the moving complex in the contracting and expanding DNA loops, respectively. In addition, we show that the highly positively supercoiled DNA generated by the EcoAI mutant is cleaved by EcoAI wild-type endonuclease much more slowly than relaxed DNA. This suggests that the topological changes in the DNA substrate associated with DNA translocation by type I restriction enzymes do not appear to be the trigger for DNA cleavage.     

Compacting DNA During the Interphase: Condensin Maintains rDNA Integrity

During mitosis, condensin is responsible for folding chromatin fibers into highly compact chromosomes, ensuring the faithful segregation of replicated chromosomes into daughter cells after each cell division. Our laboratory has unexpectedly found that condensin is capable of compacting DNA during the interphase: upon nutrient starvation, condensin is loaded to the rDNA array, leading to DNA condensation in this region. This subchromosomal DNA condensation appears to protect the integrity of the rDNA array. These observations provide the first microscopic evidence of DNA compaction by condensin outside mitosis. In addition, they show that condensin is also highly regulated during the interphase.     

Identification of a chromosome-targeting domain in the human condensin subunit CNAP1/hCAP-D2/Eg7

CNAP1 (hCAP-D2/Eg7) is an essential component of the human condensin complex required for mitotic chromosome condensation. This conserved complex contains a structural maintenance of chromosomes (SMC) family protein heterodimer and three non-SMC subunits. The mechanism underlying condensin targeting to mitotic chromosomes and the role played by the individual condensin components, particularly the non-SMC subunits, are not well understood. We report here characterization of the non-SMC condensin component CNAP1. CNAP1 contains two separate domains required for its stable incorporation into the complex. We found that the carboxyl terminus of CNAP1 possesses a mitotic chromosome-targeting domain that does not require the other condensin components. The same region also contains a functional bipartite nuclear localization signal. A mutant CNAP1 missing this domain, although still incorporated into condensin, was unable to associate with mitotic chromosomes. Successful chromosome targeting of deletion mutants correlated with their ability to directly bind to histones H1 and H3 in vitro. The H3 interaction appears to be mediated through the H3 histone tail, and a subfragment containing the targeting domain was found to interact with histone H3 in vivo. Thus, the CNAP1 C-terminal region defines a novel histone-binding domain that is responsible for targeting CNAP1, and possibly condensin, to mitotic chromosomes.     

Molecular and genetic analysis of condensin function in vertebrate cells

We engineered mutants into residues of SMC2 to dissect the role of ATPase function in the condensin complex. These residues are predicted to be involved in ATP binding or hydrolysis and in the Q-loop, which is thought to act as a mediator of conformational changes induced by substrate binding. All the engineered ATPase mutations resulted in lethality when introduced into SMC2 null cells. We found that ATP binding, but not hydrolysis, is essential to allow stable condensin association with chromosomes. How SMC proteins bind and interact with DNA is still a major question. Cohesin may form a ring structure that topologically encircles DNA. We examined whether condensin behaves in an analogous way to its cohesin counterpart, and we have generated a cleavable form of biologically active condensin with PreScission protease sites engineered into the SMC2 protein. This has allowed us to demonstrate that topological integrity of the SMC2-SMC4 heterodimer is not necessary for the stability of the condensin complex in vitro or for its stable association with mitotic chromosomes. Thus, despite their similar molecular organization, condensin and cohesin exhibit fundamental differences in their structure and function.     

Free Energy Calculations Reveal Rotating-Ratchet Mechanism for DNA Supercoil Relaxation by Topoisomerase IB and its Inhibition

Topoisomerases maintain the proper topological state of DNA. Human topoisomerase I removes DNA supercoils by clamping a duplex DNA segment, nicking one strand at a phosphodiester bond, covalently attaching to the 3′ end of the nick, and allowing the DNA downstream of the cut to rotate around the intact strand. Using molecular dynamics simulations and umbrella sampling free energy calculations, we show that the rotation of downstream DNA in the grip of the enzyme that brings about release of positive or negative supercoils occurs by thermally assisted diffusion on ratchet energy profiles. The ratchetlike free-energy-versus-rotation profile that we compute provides a model for the function of topoisomerase in which the periodic maxima along the profile modulate the rate of supercoil relaxation, while the minima provide metastable conformational states for DNA religation. The results confirm previous experimental and computational work, and suggest that relaxation of the two types of supercoils involves distinct protein pathways. Additionally, simulations performed with the ternary complex of topoisomerase, DNA, and the chemotherapeutic drug topotecan show important differences in the mechanisms for supercoil relaxation when the drug is present, accounting for the relative values of relaxation rates measured in single-molecule experiments. Good agreement is found between rate constants from tweezer experiments and those calculated from simulations. Evidence is presented for the existence of semiopen states of the protein, which facilitate rotations after the initial one, as a result of biasing the protein into a conformation more favorable to strand rotation than the closed state required for nicking of the DNA.     

A role of topoisomerase II in linking DNA replication to chromosome condensation

The condensin complex and topoisomerase II (topo II) have different biochemical activities in vitro, and both are required for mitotic chromosome condensation. We have used Xenopus egg extracts to investigate the functional interplay between condensin and topo II in chromosome condensation. When unreplicated chromatin is directly converted into chromosomes with single chromatids, the two proteins must function together, although they are independently targeted to chromosomes. In contrast, the requirement for topo II is temporarily separable from that of condensin when chromosome assembly is induced after DNA replication. This experimental setting allows us to find that, in the absence of condensin, topo II becomes enriched in an axial structure within uncondensed chromatin. Subsequent addition of condensin converts this structure into mitotic chromosomes in an ATP hydrolysis-dependent manner. Strikingly, preventing DNA replication by the addition of geminin or aphidicolin disturbs the formation of topo II-containing axes and alters the binding property of topo II with chromatin. Our results suggest that topo II plays an important role in an early stage of chromosome condensation, and that this function of topo II is tightly coupled with prior DNA replication.     

Phosphorylation of CAP-G is required for its chromosomal DNA localization during mitosis

Condensin is a 5 subunit complex that plays an important role in the structure of chromosomes during mitosis. It is known that phosphorylation of condensin subunits by cdc2/cyclin B at the beginning of mitosis is important for condensin activity, but the sites of these phosphorylation events have not been identified nor has their role in regulating condensin function. Here we identify two threonine residues in the CAP-G subunit of condensin, threonines 308 and 332, that are targets of cdc2/cyclin B phosphorylation. Mutation of these threonines to alanines results in defects in CAP-G localization with chromosomes during mitosis. These results are the first to identify phosphorylation sites within the condensin complex that regulate condensin localization with chromosomal DNA.     

Superhelicity-driven homologous DNA pairing by yeast recombination factors Rad51 and Rad54

Yeast Rad51 recombinase has only minimal ability to form D loop. Addition of Rad54 renders D loop formation by Rad51 efficient, even when topologically relaxed DNA is used as substrate. Treatment of the nucleoprotein complex of Rad54 and relaxed DNA with topoisomerases reveals dynamic DNA remodeling to generate unconstrained negative and positive supercoils. DNA remodeling requires ATP hydrolysis by Rad54 and is stimulated by Rad51-DNA nucleoprotein complex. A marked sensitivity of DNA undergoing remodeling to P1 nuclease indicates that the negative supercoils produced lead to transient DNA strand separation. Thus, a specific interaction of Rad54 with the Rad51-ssDNA complex enhances the ability of the former to remodel DNA and allows the latter to harvest the negative supercoils generated for DNA joint formation.     

Condensin but not cohesin SMC heterodimer induces DNA reannealing through protein-protein assembly

Condensin and cohesin are chromosomal protein complexes required for chromosome condensation and sister chromatid cohesion, respectively. They commonly contain the SMC (structural maintenance of chromosomes) subunits consisting of a long coiled-coil with the terminal globular domains and the central hinge. Condensin and cohesin holo-complexes contain three and two non-SMC subunits, respectively. In this study, DNA interaction with cohesin and condensin complexes purified from fission yeast was investigated. The DNA reannealing activity is strong for condensin SMC heterodimer but weak for holo-condensin, whereas no annealing activity is found for cohesin heterodimer SMC and Rad21-bound heterotrimer complexes. One set of globular domains of the same condensin SMC is essential for the DNA reannealing activity. In addition, the coiled-coil and hinge region of another SMC are needed. Atomic force microscopy discloses the molecular events of DNA reannealing. SMC assembly that occurs on reannealing DNA seems to be a necessary intermediary step. SMC is eliminated from the completed double-stranded DNA. The ability of heterodimeric SMC to reanneal DNA may be regulated in vivo possibly through the non-SMC heterotrimeric complex.     

Real‐time detection of condensin‐driven DNA compaction reveals a multistep binding mechanism

Condensin, a conserved member of the SMC protein family of ring‐shaped multi‐subunit protein complexes, is essential for structuring and compacting chromosomes. Despite its key role, its molecular mechanism has remained largely unknown. Here, we employ single‐molecule magnetic tweezers to measure, in real time, the compaction of individual DNA molecules by the budding yeast condensin complex. We show that compaction can proceed in large steps, driving DNA molecules into a fully condensed state against forces of up to 2 pN. Compaction can be reversed by applying high forces or adding buffer of high ionic strength. While condensin can stably bind DNA in the absence of ATP, ATP hydrolysis by the SMC subunits is required for rendering the association salt insensitive and for the subsequent compaction process. Our results indicate that the condensin reaction cycle involves two distinct steps, where condensin first binds DNA through electrostatic interactions before using ATP hydrolysis to encircle the DNA topologically within its ring structure, which initiates DNA compaction. The finding that both binding modes are essential for its DNA compaction activity has important implications for understanding the mechanism of chromosome compaction.     

Analysis of DNA supercoil induction by FtsK indicates translocation without groove-tracking

FtsK is a bacterial protein that translocates DNA in order to transport chromosomes within the cell. During translocation, DNA's double-helical structure might cause a relative rotation between FtsK and the DNA. We used a single-molecule technique to quantify this rotation by observing the supercoils induced into the DNA during translocation of an FtsK complex. We find that FtsK induces approximately 0.07 supercoils per DNA helical pitch traveled. This rate indicates that FtsK does not track along DNA's groove, but it is consistent with our previous estimate of FtsK's step size. We show that this rate of supercoil induction is markedly near to the ideal value that would minimize in vivo disturbance to the chromosomal supercoil density, suggesting an origin for the unusual rotational behavior of FtsK.     

Reconstitution and subunit geometry of human condensin complexes

Vertebrate cells possess two different condensin complexes, known as condensin I and condensin II, that play a fundamental role in chromosome assembly and segregation during mitosis. Each complex contains a pair of structural maintenance of chromosomes (SMC) ATPases, a kleisin subunit and two HEAT-repeat subunits. Here we use recombinant human condensin subunits to determine their geometry within each complex. We show that both condensin I and condensin II have a pseudo-symmetrical structure, in which the N-terminal half of kleisin links the first HEAT subunit to SMC2, whereas its C-terminal half links the second HEAT subunit to SMC4. No direct interactions are detectable between the SMC dimer and the HEAT subunits, indicating that the kleisin subunit acts as the linchpin in holocomplex assembly. ATP has little, if any, effects on the assembly and integrity of condensin. Cleavage pattern of SMC2 by limited proteolysis is changed upon its binding to ATP or DNA. Our results shed new light on the architecture and dynamics of this highly elaborate machinery designed for chromosome assembly.     

Chromosome condensation: packaging the genome

The packaging of centimetre long DNA molecules into compact metaphase chromosomes is essential for genome segregation in anaphase. The chromosomal condensin complex plays a crucial part in this packaging, and important new insight into condensin action in vitro and in vivo has recently been gained.     

Condensin recruitment to chromatin is inhibited by Chk2 kinase in response to DNA damage

The DNA damage checkpoint, when activated in response to genotoxic damage during S phase, arrests cells in G2 phase of the cell cycle. ATM, ATR, Chk1 and Chk2 kinases are the main effectors of this checkpoint pathway. The checkpoint kinases prevent the onset of mitosis by eliciting well characterized inhibitory phosphorylation of Cdk1. Since Cdk1 is required for the recruitment of condensin, it is thought that upon DNA damage the checkpoint also indirectly blocks chromosome condensation via Cdk1 inhibition. Here we report that the G2 damage checkpoint prevents stable recruitment of the chromosome-packaging-machinery components condensin complex I and II onto the chromatin even in the presence of an active Cdk1. DNA damage-induced inhibition of condensin subunit recruitment is mediated specifically by the Chk2 kinase, implying that the condensin complexes are targeted by the checkpoint in response to DNA damage, independently of Cdk1 inactivation. Thus, the G2 checkpoint directly prevents stable recruitment of condensin complexes to actively prevent chromosome compaction during G2 arrest, presumably to ensure efficient repair of the genomic damage.