Why do we have linear chromosomes? A matter of Adam and Eve

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1 Mutation Research 434 Ž Community address: Minireview Why do we have linear chromosomes? A matter of Adam and Eve Fuyuki Ishikawa ), Taku Naito Laboratory of Molecular and Cellular Assembly, Graduate School of Biological Information, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama , Japan Accepted 30 March 1999 Keywords: Linear chromosome; Circular chromosome; Telomere; Meiosis 1. Introduction It is usually assumed that prokaryotic cells have circular chromosomes, whereas eukaryotic cells have linear chromosomes. One of the consequences of linear chromosomes is the presence of chromosomal ends called telomeres. Simple physical ends of DNA, such as those produced by DNA double-strand breaks Ž DSB. by ionizing radiation, are genetically unstable, mutagenic, and sometimes oncogenic Žreviewed in Ref. wx. 1. Telomeres are a complex composed of telomeric DNA and a number of telomere-specific and non-specific proteins. This large molecular assembly that forms the telomeres protects the genomic ends from end-to-end fusion or exonucleolytic erosion Žreviewed in Ref. wx. 2. Due to the end-replication problem, telomeric DNA is shortened as the cell divides wx 3. In most eukaryotes, this shortening of telomeric DNA is compensated by the activity of an enzyme called telomerase that synthesizes telomeric DNA de novo wx 4. However, telomerase is strictly regulated to be inactive in most human somatic cells, and telomere lengths decline as an individual ages wx 5. This results ) Corresponding author. fishikaw@bio.titech.ac.jp in cellular senescence and cancer development due to telomere insufficiencies Že.g., Ref. wx 6, and rewx. 7. Therefore, telomeres are some- viewed in Ref. times referred to as the the Achilles heel of the chromosome wx 8. Why do we have linear chromosomes that lead to senescence and cancers, instead of circular chromosomes? In this article, we review as to what extent different chromosome configurations are conserved among different kingdoms, and propose a hypothesis to explain why this remarkable conservation has evolved. 2. Chromosome configurations of prokaryotes and eukaryotes In this article, we operatively define chromosomes as genetic materials containing house-keeping genes essential for the cell s survival that replicate synchronously with cell division to distinguish them from extra-chromosomal genetic elements, such as plasmids, bacteriophage and transposons wx 9. The recent invention of molecular biological tools to analyze large DNA structures, especially pulse field gel electrophoresis Ž PFGE., in addition to classical r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž. PII: S

2 100 F. Ishikawa, T. NaitorMutation Research tools, such as genetic linkage studies, have greatly enriched our knowledge about the chromosome configuration in different species, phyla and kingdoms. Given the very large number of species on the earth, both known and unknown, it is far from possible to make a complete catalogue of the chromosome structures existing among the diverse range of species. However, recent studies have identified the presence of several exceptional cases to the general rule that one circular chromosome is present in prokaryotic cells and more than one linear chromosome are present in eukaryotic cells. Nevertheless, these exceptional cases indeed further strengthen the notion that prokaryotes and eukaryotes have maintained circular and linear chromosomes, respectively, throughout their evolution Prokaryotes Generally, the small sizes of prokaryotic genomes Ž typically 1 10 Mb. make it feasible to carry out physical analyses, such as PFGE and total nucleotide sequencing Žreviewed in Ref. w10 x.. Genome analyses of Archea have lagged behind that of eubacteria. For example, the genome sequencing of only six archebacteria had been completed, whereas 17 eubacteria had been sequenced at the time of this review Ž All archebacteria examined so far contain one circular chromosome. Therefore, we will focus on several exceptional cases in eubacteria, where this general rule is not applied. Borrelia, Treponema and Leptospira are members of the spirochete group Ž Phylum Spirochaetae.. They are pathogenic bacteria, and Borrelia causes Lyme disease. Borrelia burgdorferi has been shown to have one 910-kb linear chromosome w11 13 x. This unique feature is conserved in the other members of the Borrelia genus w14 x. In contrast, other two closely related genera, Treponema and Leptospira, which also belong to the Spirochaetae, contain circular chromosomes w15 17 x. This specific distribution of linear chromosomes in the Borrelia genus suggests that Borrelia s linear chromosomes were evolutionarily derived from the ancestral circular chromosomes after the three genera had diverged. Streptomyces is another genus that has been shown to possess linear chromosomes w18 x. It should be noted that Streptomyces, a member of Phylum Actinomycete, is phylogenically distant from Borrelia. The closely related genus, Mycobacterium, which is within the same phylum, contains a conventional single circular chromosome w19 x, again suggesting the relatively recent origin of the linear chromosome in Streptomyces. Finally, Agrobacterium tumefaciens, a member of Proteobacteria has one 2.1-Mb linear and three 3- Mb, 450-kb and 200-kb circular replicons. It is known that at least the two 1-Mb and 3-Mb replicons contain metabolically essential genes, which classifies them as chromosomes w20 x. The conventional DNA replication mechanism does not replicate the very ends of linear DNA, because all DNA polymerases need primers for initiating synthesis. The diverse range of linear genomes solves this end-replication problem by different strategies. Adenovirus initiates the replication of its linear genome using a protein primer Žterminal protein, TP. w21 x. TP forms a covalent bond with the 5 X -OH of dnmp, and the DNA polymerase starts synthesis using this base as the first nucleotide to be incorporated. As a result, adenovirus DNA has a covalently associated TP at its 5 X -ends. On the other hand, vaccinia virus has a hairpin structure at both ends of its linear genome w22 x. One strand is continuous to the other strand, and DNA synthesis continues onto the next strand after completing one strand. A palindromic sequence is left after nicking the hairpin DNA to resolve the two daughter duplex DNAs. The terminal structures of the linear genomic DNAs of Borrelia and Streptomyces have also been reported. Streptomyces has 5 X -end associated proteins, suggesting that the telomeres are replicated by TP primers in this bacterium w23,24 x. In contrast, Borrelia has hairpin structures with 26-bp inverted repeats at both telomeres, suggesting that its telomeres are replicated in a way similar to vaccinia virus w25 x. These studies indicate that at least three prokaryotic genera possess linear chromosomes, instead of circular ones. However, the appearance of linear chromosomes in prokaryotes seems rather sporadic. First, these three genera are distantly related to each other. Second, closely related genera belonging to the same phylum contain conventional circular chromosomes. Finally, the solutions for the end-replication problems differ between Borrelia and Strep-

3 F. Ishikawa, T. NaitorMutation Research tomyces. Accordingly, it is suggested that prokaryote linear chromosomes have not been inherited directly from one ancient prokaryote that had linear chromosomes. Instead, they most likely have developed recently from the circular chromosomes of an ancestor species. Therefore, it may be concluded that the prokaryote genomes have been maintained phylogenically in circular forms Eukaryotes Eukaryotes contain larger genomes than prokaryotes Ž typically larger than 10 Mb.. Accordingly, in many cases, the chromosomes can be visualized by microscopy to analyze the gross structures. However, large chromosome sizes are a disadvantage in another respect, since it is usually difficult to construct a physical map of the genome. As will be reviewed here, many reports have been published showing the presence of circular chromosomes. However, the evidence for covalently linked circular chromosomes is not available in most cases. To avoid possible confusion, circular chromosomes judged solely on morphological criteria will be called ring chromosomes in this review. Circular chromosomes have been reported both in budding yeast w26,27x and fission yeast w28,29 x. They were isolated spontaneously or artificially, and have been shown to be circular by either a genetic or a physical approach. In each case, only one circular chromosome was identified, and it was unstable mitotically and meiotically. As there are many opportunities to examine karyotypes in a variety of medical settings, many cases of ring chromosomes have been reported to be associated with a variety of clinical manifestations. In most cases, the ring chromosomes have been found in somatic cells, either normal or cancerous, and either constitutively or in mosaicism. However, few notable cases in which one ring chromosome had apparently been inherited from one of the parents who also had the same ring chromosomes have been reported Že.g., Ref. w30 x.. In these cases, the parents generally showed mosaic ring chromosomes, suggesting that an individual who has a ring chromosome constitutively is infertile. There has been no report describing the inheritance of more than one ring chromosomes. These results have suggested that in some rare cases, a single ring chromosome may be normally segregated in meiosis, and fertilized. In summary, circular or ring chromosomes have been found sporadically in eukaryotes. With some rare exceptions, they are not usually inherited. However, there have been no reports describing eukaryotic cells having more than one circular or ring chromosome that have been meiotically transmitted. These results suggest that ring chromosomes face some difficulty in sexual reproduction. Since mitochondrial DNAs are circular in most eukaryotes, chromosome circularity itself is obviously not incompatible with inheritance. Circular chromosomes may be incompatible with a process specific to sexual reproduction, such as meiosis. This section has shown that two chromosome configurations, circular and linear, are remarkably conserved in prokaryotes and eukaryotes, respectively. In an evolutionary sense, linear chromosomes require extra energy to maintain intact telomeres, which is not a requirement for circular chromosomes. This reasoning suggests that there must be some advantages to eukaryotes that have been acquired in a trade-off for this extra burden. One of the most direct experiments to test this hypothesis would be to construct a eukaryotic cell that maintains its genome in a circular form, and to see what biological functions this cell has lost. However, until recently, there has been no report that describes the existence of eukaryotic cells that maintain completely circular genomes. 3. ATM family genes and telomeres Telomeres are comprised of many components to accomplish its functions. Proteins involved in telomere maintenance are now being studied in some detail, especially in simple eukaryotic cells such as yeast Žreviewed in Ref. w31 x.. One group of interesting proteins thus identified is the ATM family. The Saccharomyces cereõisiae TEL1 gene was originally identified by screening for mutants with short telomw32 x. When this gene was cloned, it ere phenotypes turned out to have a significant level of homology with the human ATM gene, whose mutations cause the hereditary disease, ataxia telangiectasia Ž A-T.

4 102 F. Ishikawa, T. NaitorMutation Research w33,34 x. Interestingly, telomeres in A-T cells also showed excessive shortening w35,36 x, suggesting that the ATM family genes are involved in telomere maintenance in all cells from yeast to human. There is at least more than one member of the ATM family genes present in one species w37 x. The budding yeast Saccharomyces has TEL1 and MEC1 genes, and human has ATM, ATR and the relatively distant member DNA-PK. All these proteins have a PI3 Ž phosphatidylinositol 3. -kinase-like domain at the C-termini, yet they have protein kinase activity. Fission yeast, Schizosaccharomyces pombe, also has two ATM family genes, named rad3 q and tel1 q w x q q 38,39. The mutant defective for rad3 or tel1 shows moderate to minimal telomere shortenings w39,40 x. However, when both ATM family genes were mutated, there was an additive effect, and the fission yeast chromosomes essentially lost all telomw39 x. Thus, the ATM family genes are ere sequences redundant but essential for stable telomere maintenance. The rad3 tel1 double mutant cells grow very slowly with a low viability, and showed aberrantly irregular colony shapes, as expected for cells suffering from extensive telomere shortening. However, derivative cells that showed apparently normal colony shapes appeared spontaneously among these double mutants at a relatively high frequency. Surprisingly, these derivative cells contain three self-circularized chromosomes Žfission yeast contains three linear chromosomes. w39 x. This case is the first report describing the existence of eukaryotic cells that maintain the genome exclusively in circular forms. Previously, it had been postulated that eukaryotic cells do not have circular chromosomes because if an odd number of crossing-over events occurs between the two sister chromatids by SCE, this would result in the formation of dicentric circular chromosomes. Thus, formed circular dimer chromosomes would be eventually broken during mitosis by the two spindles pulling them apart Že.g., see discussions in Refs. w41,42 x.. Since yeasts undergo efficient homologous recombination and SCEs, we would expect that complete circular genomes in yeast should be highly unstable and lead to cell death. Indeed, the fission yeast rad3 tel1 mutant with the three circular chromosomes showed anaphase bridges and some degree of aneuploidy Ž Naito and Ishikawa, unpublished.. However, the fact that this mutant grew well mitotically as a mass suggests that the SCE of dicentric circular chromosomes may have a relatively small effect. Indeed, SCE happens in prokaryotic cells to produce circular dimers. In Escherichia coli, these circular dimers are known to be resolved by both the reca-independent resolvases, XerC and XerD, that act on a specific locus called dif located at the replication terminus, and the reca-dependent recombination pathway Žreviewed in Refs. w43,44 x.. In higher eukaryotes, genomic DNA is organized into multiple loops by tight association of matrix-associated region Ž MAR. on DNA with nuclear scaffolds. In a topological sense, each loop can be assumed microscopically to be a closed circle. Therefore, closed circular oligomers may also be formed by SCE in linear eukaryotic genomes, making the hypothesis that the circular dimer formation prohibits circular chromosomes in eukaryotes unlikely. Eukaryotes may have mechanisms similar to XerCD and reca-dependent recombination in E. coli to resolve these microscopic oligomers. Indeed, RAD51-deficient chicken cells, the eukaryote recahomologue, are shown to be arrested in G2rM phase and to accumulate chromosome breaks, suggesting the possible involvement of the Rad51 protein in resolving SCE-intermediates w45 x. 4. Sister chromatid exchanges SCEs and circular dimer formation 5. Meiosis and telomeres Telomeres perform a number of important functions in different biological situations. Recently, the role of the telomeres in meiosis has come into light Žreviewed in Ref. w46 x.. The first hint came from the cytological observation that telomeres are closely clustered with each other at a specific stage of meiosis. There have been ample observations that telomeres and centromeres are positioned asymmetrically in nuclei Žreviewed in Ref. w47 x.. In mitotic interphase, the centromeres have a tendency to cluster around the centriole, presumably reflecting the

5 F. Ishikawa, T. NaitorMutation Research association between these two structures during the last anaphase. This centromere clustering is named the Rabl w48x orientation after the German scientist who first described it. However, during meiotic prophase, this relative distribution of centromeres and telomeres is reversed: In the leptotenerzygotene stage, telomeres, instead of centromeres, are clustered at the inner surface of the nuclear envelope. The centromeres are distributed randomly in the nucleus at this stage. This polarized chromosomal distribution is called the bouquet arrangement w49 x, and is found during meiosis in many species Žre- w50 x.. The functional significance of viewed in Ref. this peculiar conformation has been recently revealed. In fission yeast, two haploid cells of opposite mating types conjugate to produce diploid cells Ž karyogamy., and enter meiosis Ž zygotic meiosis.. Immediately after karyogamy, the fused nucleus forms an elongated shape, called a horse-tail w51 x. Using time-elapsed image recording, the fused horse-tail nuclei were found to undergo a dynamic to-and-fro oscillating movement w52 x. Moreover, by a combination of telomere-specific fluorescence in situ hybridization Ž FISH. and immunostaining of the spindle pole body ŽSPB, a centriole-counterpart in yeast., it was shown that six telomeres of the three fission yeast chromosomes are closely associated with SPB, and they lead the front edge of this horse-tail movement. In fission yeast, the bouquet arrangement is established by an association between the telomeres and SPB. In this way, the microtubule enucleated from the SPB promotes the dynamic nuclear movement by pulling the telomeres and dragging the chromosomes behind as a mass Žreviewed in Ref. w53 x.. Recently, three additional genetic studies have further indicated the importance of telomeres in the meiotic process. Telomere DNA consists of wellconserved G-rich simple tandem repeats. Telomere DNA-specific binding proteins are known to exist in several species. These include TRF1 and TRF2 in human w54 x, Rap1 in budding yeast w55 x, and taz1p in fission yeast w56 x. Fission yeast mutants defective for taz1 q failed to form the telomere clustering at the horse-tail stage of pre-meiosis, and showed reduced spore viability w57,58 x. Taz1p is presumably involved in the SPB-telomere association w53 x. The rad3 tel1 fission mutant with the three circular chromosomes was examined for spore viability after azygotic meiosis w39 x. In this case, diploid cells derived from two haploid cells harboring circular chromosomes produced no viable spores, a phenotype more profound than that of the taz1 mutant. These studies clearly indicated that telomeres are essential for a productive meiotic process. 6. How telomeres are essential for meiosis Several scenarios can be proposed to explain why functional telomeres are essential for meiosis. Meiosis consists of two successive cell divisions, called meiosis I and meiosis II. Meiosis II is similar to mitotic cell division, but meiosis I is unique. Meiosis is a process that produces four haploid cells from one diploid cell. Meiosis I is responsible for this reduction of ploidy by segregating the two homologous chromosomes to the two daughter cells. To accomplish this reductional segregation, each pair of homologous chromosomes needs to be paired before the onset of meiosis I Žreviewed in Ref. w59 x.. Homologous chromosome pairing has another important role in inducing homologous recombination between the two homologues. This homologous recombination shuffles the two alleles originally derived from different individuals Ž father and mother., and ensures that the haploid cells contain chimeric genetic information. Moreover, the recombination and segregation are interdependent, since the covalent associations formed by the recombinational Holiday junction between the two homologues Ž chiasmata. are thought to be essential for stable homologue pairing, and ensuring proper segregation. Therefore, homologue-pairing is at the heart of the mechanism of meiosis. Circular chromosomes potentially undergo more than one pathway during meiosis, and in all cases, they have very small probabilities of proper segregation Ž Fig. 1.. In normal meiotic prophase, linear chromosomes gather together by telomere-clustering Ž Fig. 1A.. Telomere associations of homologous chromosomes may help the homologue pairing by aligning the two chromosomes that are now tethered at both ends. After successful pairing, homologous recombination occurs between the two homologues,

6 104 F. Ishikawa, T. NaitorMutation Research Ž. Ž. Fig. 1. Meiosis I of linear chromosomes A and possible pathways in meiosis I of circular chromosomes B. For details, see the text.

7 F. Ishikawa, T. NaitorMutation Research and this covalent association further contributes to the stable chromosome pairing. In anaphase I, the Holiday junctions are resolved and the two homologues are segregated to different daughter cells. In contrast, circular chromosomes may undergo several different pathways Ž Fig. 1B.. As circular chromosomes lack functional telomeres, two homologues cannot be positioned in proximity. In this case, no homologue pairing and recombination occur, and the homologues are randomly segregated to daughter cells Ž pathway 1.. Two homologues may be positioned closely by chance, and somehow may pair and undergo recombination Ž pathway 2.. However, if an odd number of crossing-over events occurs between two homologues, this results in the formation of dicentric circular chromosomes Ž pathway 3.. If resolvase fails to resolve this form into monomers, the dicentric circle enters anaphase. When the spindles of different origins attach to each of the two kinetochores, the chromosome will be pulled apart and tear Ž pathway 4.. When a common spindle attaches to both of the two kinetochores, the chromosome is segregated to only one cell, with the other cell receiving no homologue. In either case, daughter cells will lose a significant amount of genetic information. When an even number of crossing-over events occurs Ž pathway 7., or the dicentric circles are resolved into monomeric circles Ž pathway 6., the two homologues may be segregated properly to the two daughter cells. However, even in this case, it is not known if spindles correctly attach to the kinetochores of chromosomes that have not been associated with telomeres during the meiotic prophase. Overall, the chance that one particular circular chromosome is segregated properly in meiosis I is very small. All eukaryotes contain more than one and usually many chromosomes. The chance, that one daughter cell will have all chromosomes properly segregated in circular forms, is the multiple of these small probabilities for each circular chromosome, and should be negligible. In conclusion, there is essentially no chance that all circular chromosomes are properly segregated during meiosis. This essential role of telomeres in accomplishing reductional chromosome segregation in meiosis must be the major reason that linear chromosomes are strikingly conserved in eukaryotes, which are characterized by the presence of sexual reproduction in most cases. 7. Conclusion We have stated that linear chromosomes are essential for productive meiosis. Meiosis Ža mechanism to generate haploid cells. is a prerequisite for shuffling the genetic information between individuals. It has been proposed that the production of genetically diverse offspring is advantageous in an ever-changw60 x. Indeed, the num- ing or saturated environment ber of absolutely asexual eukaryotic organisms is very small Žreviewed in Refs. w61,62 x., and the conservation of the potential of sexual reproduction seems to be as strong as the conservation of linear chromosomes in eukaryotes. Recent studies have indicated that chromosome linearity is important for meiosis, and we would like to propose that the correlation between sex and linear chromosomes is based on a mechanistic reason, and not on a superficial parallelism. Once upon a time, two groups of living creatures emerged from a common ancestor. One group decided to maintain genomes in circular forms, because this form is more economical without the need to maintain telomeres. However, the progeny of this group Ž Bacteria. is not able to exchange genomic information by meiosis and fertilization, and thus, needs to grow faster and keep the genome size as small as possible. The other group decided to maintain the genomes in linear forms. Although this strategy requires extra energy to maintain telomeres, these organisms have enjoyed the dynamic flow of genomic information by sexual reproduction. This process has allowed this group Ž Eukaryota. the chance to produce a variety of offspring. Accordingly, eukaryotes have complicated systems, and grow less rapidly than prokaryotes. Acknowledgements We thank E.A. Kamei Ž Gunma University. and H. Niki Ž Kumamoto University. for critical reading of and comments on the manuscript. The excellent secretarial works of F. Nishizaki, K. Saito and K. Yokoyama are acknowledged. This work was supported by a grant-in-aid from the Organization for Pharmaceutical Safety and Research, Japan.

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