Coming undone: How to untangle a chromosome

Cell, Vol. 77, 955-957,

July 1, 1994, Copyright

0 1994 by Cell Press

Minireview

Coming Undone: How to Untangle a Chromosome Connie Holm Department of Pharmacology and Division of Cellular and Molecular University of California, San Diego La Jolla, California 92093-0651

Medicine

Chromosome segregation is one of the most treacherous acts in the life of a cell. Assuming the DNA has replicated perfectly (a Herculean task in itself), the cell must precisely segregate many pairs of chromosomes, mechanically hauling them across the cytoplasm without causing structural damage to any of them. While the elegance of the mitotic spindle and related motor proteins has been appreciated for many years, only recently has it become clear that crucial feats must be performed by the chromosomes themselves. Most important, daughter DNA molecules must be put into shape for segregation. The overview of this process is revealed in cytological studies of sister chromatids (the DNA-protein complex of the two progeny copies of a single DNA molecule). When chromosomes condense at the beginning of mitosis, the two replicated sister chromatids initially appear to form a single long cylinder. As condensation proceeds, the single cylinder resolves into two thinner cylinders that are closely apposed along their entire length (Sumner, 1991). At this point, sister chromatids are ordinarily transported to opposite poles of the cell by the mitotic apparatus. Remarkably, however, these individualized cylinders can separate from one another without the aid of microtubules. If the microtubule attachment point of one of the sister chromatids is destroyed, the sisters will nonetheless separate from one another on schedule, although they will not move very far (McNeil1 and Bern% 1961). Thus, it has been proposed that there is adistinction between chromosome separation, in which the two sister chromatids are resolved from one another, and chromosome segregation, in which the separated sisters are mechanically transported to opposite sides of the cell (Miyazaki and Orr-Weaver, 1994). Whatever mechanism separates sister chromatids, it must overcome two important problems. The first is that the chromosomes are very long and susceptible to tangling; each tangle must be removed to allow sister chromatids to separate without damage. The second problem is that chromosomes are structurally very similar to one another. Thus, the cell must keep sister chromatids close together to facilitate identification of those chromatids that should be segregated from one another during mitosis. The problems of disentangling sister chromatids while nonetheless keeping them in physical proximity are the subjects of this minireview. Eukaryotic Chromosomes Are Tangled after DNA Replication Tangling of sister chromatids is a problem of enormous proportions. Because the two strands of DNA are interwound about one another (a plectonemic coil), direct

DNA replication without concomitant disentangling would lead to daughter molecules that are wound around one another thousands of times (Figure 1). Studies in SV40 reveal that most of the potential tangles are resolved by topoisomerases during the process of DNA replication (Sundinandvarshavsky, 1981; Weaveret al., 1985). However, it appears that the topoisomerases are sterically excluded as some of the replication forks approach one another. Thus, when replication forks collide, a few intertwinings between the progeny DNA molecules are often introduced. In eukaryotic chromosomes, tremendous size and numerous replication forks probably lead to multiple intertwinings. Although one might imagine that intertwinings between linear chromosomes could simply slide off the ends of the chromosomes during segregation, only those intertwinings on very short chromosome arms appear able to do so (Spell and Holm, 1994). If sister chromatids are not disentangled prior to chromosome segregation, the results for the cell are disastrous. In contrast with many other essential cellular processes, there appears to be no regulatory checkpoint for DNA untangling. When untangling is prevented by mutation or treatment with drugs, chromosome segregation nonetheless proceeds. This ill-advised segregation leads to chromosome breakage, nondisjunction, and cell death (Holm et al., 1985, 1989; Uemura and Yanagida, 1986; Uemura et al., 1987; Downes et al., 1991; Spell and Holm, 1994). Given these dire consequences, it was initially surprising to discover that the cell postpones the job of disentangling its chromosomes until the time of mitosis (Uemura et al., 1987; Holm et al., 1989). The explanation for this per-

Figure

1, DNA Replication

Produces

intertwined

Chromosomes

As replication proceeds (top), the proteins required to resolve ings may be sterically excluded when the replication forks one another (middle), leading to tangled sister molecules Only a small segment of chromosomal DNA is shown here. from Varshavsky et al. (1983).

interhvinapproach (bottom). Adapted

Cell 956

Figure 2. Sister Chromatids regation

Are Untangled

during

Chromosome

Seg

Replicated sister chromatids (top) undergo final untangling at the time of chromosome segregation (middle) via the action of DNA topo II (closed triangles), before being segregated to opposite poles (bottom). Direction of chromosome segregation is indicated by open arrows.

plexing observation probably lies with the properties of the enzyme responsible for untangling sister chromatids. Chromosomes Must Be Untangled to Be Effectively Segregated Chromosomal intertwinings are resolved by the activity of DNA topoisomerase II (topo II), which acts by making a double-strand cut in one sister DNA molecule, passing the other double strand through the cut, and then resealing the cut. Unfortunately, topo II is a rather unintelligent enzyme, acting either to tangle or disentangle DNA molecules with equal enthusiasm. When DNA concentrations are high in vitro, topo II preferentially entangles circular DNA molecules; when concentrations are low, it preferentially disentangles them (Kreuzer and Cozzarelli, 1980). In vivo, it appears that the reaction catalyzed by topo II is driven toward disentanglement as the result of two processes: chromosome condensation and chromosome movement. Topo II begins untangling sister chromatids during chromosome condensation, when the cell packages DNA compactly for efficient travel across the cytoplasm. Normal condensation is inhibited by depletion of topo II in vitro (Wood and Earnshaw, 1990; Adachi et al., 1991), and topo II is required in vivo for the additional condensation (hypercondensation) that occurs when cells are treated with microtubule inhibitors (Uemura et al., 1987). The requirement for topo II during condensation is probably a result of the reversibility of the untangling reaction. As long as sister molecules are extended, topo II can sequentially tangle them, untangle them, and then tangle them once more. Once condensation of each DNA molecule into its individual cylinder begins, however, the products of the reaction will be separated by the process of compaction. Thus, much of the untangling of sister chromatids probably occurs during chromosome condensation. The job of untangling sister chromatids cannot be completed until the time of anaphase (chromosome segregation itself; Figure 2). When yeast cells are arrested with microtubule inhibitors just prior to anaphase (i.e., when

they are given extra time to resolve chromosome tangles), they still require topo II activity to segregate their chromosomes successfully (Uemura et al., 1987; Holm et al., 1989). This result is particularly dramatic in fission yeast because it is possible to visualize the centromeres being pulled out from the bulk of the chromosomal material, which itself is left behind in a tangled mass (Funabiki et al., 1993). Experiments with cycling Xenopus extracts reveal that this result is not specific to cells arrested with microtubule inhibitors; topo II is required to untangle sister chromatids even after prolonged incubation at metaphase (Shamu and Murray, 1992). Although these results do not demonstrate whether the final untangling of sister chromatids is facilitated by the action of microtubules, they can once again be rationalized in terms of the reversibility of the reaction catalyzed by topo II. As the disentangled products are separated, either by the mitotic apparatus or by some other mechanism, the untangling reaction goes to completion. Although the mechanics of condensation and segregation are sufficient to explain how sister chromatids can be disentangled by a reversible enzyme, a puzzle nonetheless remains. The forces in metaphase are approximately equal to the forces in anaphase (Nicklas, 1988; Alexander and Rieder, 1991), so why do sister chromatids not float apart as soon as they are pulled by the mitotic apparatus? Surely this pulling should be enough to give topo II sufficient impetus to separate sister chromatids. The answer appears to be that sister chromatids are held together by other mechanisms. Sister Chromatids Are Held Together until the Time of Chromosome Segregation If sister chromatids were completely separated from one another prior to the time of chromosome segregation, the structural similarity of chromosomes would make it difficult for the cell to identify replicative sisters. (Unlike meiotic cells, mitotic cells appear to have no specialized pairing process prior to segregation.) Maintaining a connection between replicated sister centromeres provides a clever way for the mitotic cell to ensure that it segregates its chromosomes correctly. Experiments with meiotic cells reveal that tension is required to maintain a stable connection between microtubules and a centromere (Nicklas and Koch, 1989). Thus, if sister chromatids remain attached to one another, their attachment to microtubules should be stable only when the sister centromeres are oriented toward opposite poles of the cell. In summary, if the physical proximity of sister chromatids is maintained throughout metaphase, the properties of microtubule attachment should ensure that sister chromatids are segregated to opposite poles. A variety of mechanisms have been proposed to maintain the proximity of sister chromatids until the time of anaphase. For example, one possibility was that latereplicating centromeric DNA might serve to hold sister chromatids together. Doubt was cast on this hypothesis when it was not possible to detect delayed replication of centromeric DNA in human chromosomes (Comings, 1966). More recently, density shift experiments in Saccharomyces cerevisiae demonstrated conclusively that

Minireview 957

centromeric DNA is fully replicated well before the time of chromosome segregation (McCarroll and Fangman, 1988). An alternative hypothesis is that residual intertwinings between sister chromatids maintain the connection between the sisters (Murray and Szostak, 1985). Under this scenario, intertwinings would be more than an artifact of incomplete untangling; in addition, theywould perform the essential function of holding sister chromatids together until anaphase. This model is attractive because it explains the results of several experiments and because it postulates no new properties of replicated chromosomes. However, this hypothesis is at odds with some experimental evidence from S. cerevisiae. Although in situ hybridization reveals that sister minichromosomes are held in close physical proximity (Guacci et al., 1994), these sister chromatids are clearly not intertwined in cells arrested just prior to chromosome segregation (Koshland and Hartwell, 1987). Furthermore, small natural chromosomes remain intact when segregated in the absence of topo II activity; this observation suggests that small chromosomes do not carry stable intertwinings that could oppose mitotic spindle forces (Spell and Holm, 1994). It remains possible, however, that intertwinings provide an initial state that facilitates the formation of a second, more stable attachment between sister chromatids. Recent results suggest the possibility that protein linkages may mediate stable attachment between sister chromatids. This idea is especially attractive in light of observations that it is general proteolysis, rather than specific degradation of cyclin, that leads to the metaphase-anaphase transition (Holloway et al., 1993). Thus, an economical model is that sister chromatids are held together in the centromere region by linking proteins that are themselves susceptible to proteolysis. When proteolysis is initiated, the linking proteins would be cleaved, and anaphase chromosome movements would begin. The identity of the putative linking proteins is by no means clear, but numerous candidates exist. They include the gene products identified by a large number of sister chromatid cohesion mutants and by antibodies such as the INCENP antibodies (Cooke et al., 1987; for review see Miyazaki and OrrWeaver, 1994). With these candidates, plus the rapidly unfolding identification of yeast centromere-binding proteins, the future looks bright for untangling the mystery of sister chromatid attachment in mitosis.

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