CTF7 (chromosome transmission fidelity) gene in budding yeast encodes an essential protein that is required for high-fidelity chromosome transmission and contains regions of identity conserved from yeast to man. ctf7 mutant cells arrested prior to anaphase onset contain separated sister chromatids. Thus, Ctf7p is essential for cohesion. Cohesion is established during S phase and then maintained until mitosis. However, Ctf7p activity is required only during S phase, suggesting that Ctf7p functions in the establishment of cohesion. In addition, ctf7 genetically interacts with DNA metabolism mutations pol30 (PCNA) and ctf18 (an RF-C like protein) and ctf7 temperature sensitivity and chromosome loss are rescued by high levels of POL30. These findings provide the first evidence that links the establishment of sister chromatid cohesion to the DNA replication machinery and suggest that the assembly of cohesion (and possibly condensation) complexes are coupled to PCNA-dependent DNA replication. The analysis of Ctf7p also reveals an important connection between sister chromatid cohesion, spindle integrity and the spindle assemby checkpoint.

Abstract. Most models of mitotic congression and segregation assume that only poleward pulling forces occur at kinetochores. However, there are reports for several different cell types that both mono-oriented and bi-oriented chromosomes oscillate toward and away from the pole throughout mitosis. We used new methods of high resolution video microscopy and computer-assisted tracking techniques to measure the positions over time of individual kinetochores with respect to their poles during mitosis in living newt lung cells. The results show that kinetochores oscillate throughout mitosis when they are tethered to spindle poles by attachment to the plus-ends of kinetochore microtubules (kMTs). Oscillations were not sinusoidal. Instead, kinetochores abruptly (as quick as 6 s or less) switched between persistent (,,ol.5 min average duration) phases of poleward (P) and away from the pole (AP) movement. This kinetochore "directional instability" was a property of motility at the plus-ends of kMTs since fluorescent marks on the lattice of kMTs have previously been observed to exhibit only relatively slow P movement. Each P and AP phase consisted of one or a few constant velocity domains (•1.7/zm/min average velocity). Velocities of P and AP phases were similar from prometaphase through mid-anaphase. Kinetochores occasionally switched to an indeterminant (N) phase of no or confused motion, which was usually brief compared to the durations of P and AP phases. Net chromosome displacements that occurred during congression to the equator or poleward movement during anaphase were primarily generated by differences in the durations and not the velocities of P and AP movements. Careful analysis of centromere deformation showed that kinetochore P movement produced pulling forces while kinetochore AP movement produced pushing forces. These data show that kinetochore directional instability is fundamental to the processes of chromosome congression and segregation. We argue that tension at the kinetochore attachment site is a key factor which controls the switching between P and AP phases of kinetochore motion.INETOCHORES in association with kinetochore microtubules (kMTs) ~ are responsible for the attachment of chromosomes to spindle poles, chromosome congression to the spindle equator during metaphase, and segregation to the spindle poles during anaphase in mitotic animal cells (for review see Salmon, 1989b;Rieder, 1991; McIntosh and Pfarr, 1991). Kinetochores become tethered to polar microtubules (MTs) by capturing and stabilizing the dynamically instable MT plus-ends (Pickett-Heaps et al., 1982;Rieder, 1982;Hayden et al

Abstract. We have used time-lapse digital-and videoenhanced differential interference contrast (DE-DIC, VE-DIC) microscopy to study the role of dynein in spindle and nuclear dynamics in the yeast Saccharomyces cerevisiae. The real-time analysis reveals six stages in the spindle cycle. Anaphase B onset appears marked by a rapid phase of spindle elongation, simultaneous with nuclear migration into the daughter cell. The onset and kinetics of rapid spindle elongation are identical in wild type and dynein mutants. In the absence of dynein the nucleus does not migrate as close to the neck as in wild-type cells and initial spindle elongation is confined primarily to the mother cell. Rapid oscillations of the elongating spindle between the mother and bud are observed in wild-type cells, followed by a slower growth phase until the spindle reaches its maximal length. This stage is protracted in the dynein mutants and devoid of oscillatory motion. Thus dynein is required for rapid penetration of the nucleus into the bud and anaphase B spindle dynamics. Genetic analysis reveals that in the absence of a functional central spindle (ndcl), dynein is essential for chromosome movement into the bud. Immunofluorescent localization of dynein-13-galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies and the cell cortex; with spindle pole body localization dependent on intact microtubules. A kinetic analysis of nuclear movement also revealed that cytokinesis is delayed until nuclear translocation is completed, indicative of a surveillance pathway monitoring nuclear transit into the bud.

From the time of DNA replication until anaphase onset, sister chromatids remain tightly paired along their length. Ctf7p/Eco1p is essential to establish sister-chromatid pairing during S-phase and associates with DNA replication components. DNA helicases precede the DNA replication fork and thus will first encounter chromatin sites destined for cohesion. In this study, I provide the first evidence that a DNA helicase is required for proper sister-chromatid cohesion. Characterizations of chl1 mutant cells reveal that CHL1 interacts genetically with both CTF7/ECO1 and CTF18/CHL12, two genes that function in sisterchromatid cohesion. Consistent with genetic interactions, Chl1p physically associates with Ctf7p/Eco1p both in vivo and in vitro. Finally, a functional assay reveals that Chl1p is critical for sister-chromatid cohesion. Within the budding yeast genome, Chl1p exhibits the highest degree of sequence similarity to human CHL1 isoforms and BACH1. Previous studies revealed that human CHLR1 exhibits DNA helicaselike activities and that BACH1 is a helicase-like protein that associates with the tumor suppressor BRCA1 to maintain genome integrity. Our findings document a novel role for Chl1p in sister-chromatid cohesion and provide new insights into the possible mechanisms through which DNA helicases may contribute to cancer progression when mutated.

CTF7/ECO1 is an essential yeast gene required for the establishment of sister chromatid cohesion. The findings that CTF7/ECO1, POL30 (PCNA), and CHL12/CTF18 (a replication factor C [RFC] homolog) genetically interact provided the first evidence that the processes of cohesion establishment and DNA replication are intimately coupled-a link now confirmed by other studies. To date, however, it is unknown how Ctf7p/Eco1p function is coupled to DNA replication or whether Ctf7p/Eco1p physically associates with any components of the DNA replication machinery. Here, we report that Ctf7p/Eco1p associates with proteins that perform partially redundant functions in DNA replication. Chl12p/Ctf18p combines with Rfc2p to Rfc5p to form one of three independent RFC complexes. By chromatographic methods, Ctf7p/Eco1p was found to associate with Chl12/Ctf18p and with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. The association between Ctf7p/Eco1p and this RFC complex is biologically relevant in that (i) Ctf7p/Eco1p cosediments with Chl12p/Ctf18p in vivo and (ii) rfc5-1 mutant cells exhibit precocious sister separation. Previous studies revealed that Rfc1p or Rad24p associates with Rfc2p to Rfc5p to form two other RFC complexes independent of Ctf18p-RFC complexes. These Rfc1p-RFC and Rad24p-RFC complexes function in DNA replication or repair and DNA damage checkpoint pathways. Importantly, Ctf7p/Eco1p also associates with Rfc1p and Rad24p, suggesting that these RFC complexes also play critical roles in cohesion establishment. The associations between Ctf7p/Eco1p and RFC subunits provide novel evidence regarding the physical linkage between cohesion establishment and DNA replication. Furthermore, the association of Ctf7p/Eco1p with each of three RFC complexes supplies new insights into the functional redundancy of RFC complexes in cohesion establishment.From the time of DNA replication until the onset of anaphase, sister chromatids remain tightly paired along their length (15,46,55). Cytological and molecular studies of cohesion factors reveal that this sister chromatid pairing, or cohesion, is an essential component of bipolar spindle formation, chromosome segregation, cell cycle progression, and doublestrand-break repair (25,49,50). Studies performed with numerous cell systems reveal that defects in cohesion between sister chromatids result in missegregation of both sisters to one daughter cell and cell death (16,23,31,35,51,52,57). In humans, chromosome missegregation allows for phenotypic expression of recessive mutations in tumor suppression and cancer-related growth control genes (29, 65).Three classes of proteins are required for sister chromatid cohesion and proper chromosome segregation. Structural cohesion proteins (or cohesins) provide the glue that maintains sister chromatid pairing from G 1 /S to anaphase onset. In budding yeast, the cohesins include Smc1p, Smc3p, Mcd1p/Scc1p, Scc3p/Irr1p, and Pds5p (16,19,28,35,42,52,57). Recent data revealed that the structural cohesins form a ring. This ring structure is thought to hold sister chr...

Background: Roberts syndrome (RBS) is a rare genetic disorder characterized by craniofacial abnormalities, limb malformation, and often severe mental retardation. RBS arises from mutations in ESCO2 that encodes an acetyltransferase and modifies the cohesin subunit SMC3. Mutations in SCC2/NIPBL (encodes a cohesin loader), SMC3 or other cohesin genes (SMC1, RAD21/ MCD1) give rise to a related developmental malady termed Cornelia de Lange syndrome (CdLS). RBS and CdLS exhibit overlapping phenotypes, but RBS is thought to arise through mitotic failure and limited progenitor cell proliferation while CdLS arises through transcriptional dysregulation. Here, we use the zebrafish regenerating fin model to test the mechanism through which RBS-type phenotypes arise. Results: esco2 is up-regulated during fin regeneration and specifically within the blastema. esco2 knockdown adversely affects both tissue and bone growth in regenerating fins-consistent with a role in skeletal morphogenesis. esco2-knockdown significantly diminishes cx43/gja1 expression which encodes the gap junction connexin subunit required for cell-cell communication. cx43 mutations cause the short fin (sof b123 ) phenotype in zebrafish and oculodentodigital dysplasia (ODDD) in humans. Importantly, miR-133-dependent cx43 overexpression rescues esco2-dependent growth defects. Conclusions: These results conceptually link ODDD to cohesinopathies and provide evidence that ESCO2 may play a transcriptional role critical for human development. Developmental Dynamics 245:7-21, 2016. V C 2015 Wiley Periodicals, Inc.

Microtubules (MTs) in newt mitotic spindles grow faster than MTs in the interphase cytoplasmic microtubule complex (CMTC), yet spindle MTs do not have the long lengths or lifetimes of the CMTC microtubules. Because MTs undergo dynamic instability, it is likely that changes in the durations of growth or shortening are responsible for this anomaly. We have used a Monte Carlo computer simulation to examine how changes in the number of MTs and changes in the catastrophe and rescue frequencies of dynamic instability may be responsible for the cell cycle dependent changes in MT characteristics. We used the computer simulations to model interphase-like or mitotic-like MT populations on the basis of the dynamic instability parameters available from newt lung epithelial cells in vivo. We started with parameters that produced MT populations similar to the interphase newt lung cell CMTC. In the simulation, increasing the number of MTs and either increasing the frequency of catastrophe or decreasing the frequency of rescue reproduced the changes in MT dynamics measured in vivo between interphase and mitosis.

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