Formation, maintenance and consequences of the imprint at the mating-type locus in fission yeast

Mating-type switching in the fission yeast Schizosaccharomyces pombe is initiated by a strand-specific imprint located at the mating-type (mat1) locus. We show that the imprint corresponds to a single-strand DNA break (SSB), which is site- but not sequence-specific. We identified three novel cis-acting elements, involved in the formation and stability of the SSB. One of these elements is essential for a replication fork pause next to mat1 and interacts in vivo with the Swi1 protein. Another element is essential for maintaining the SSB during cell cycle progression. These results suggest that the DNA break appears during the S-phase and is actively protected against repair. Consequently, during the following round of replication, a polar double-strand break is formed. We show that when the replication fork encounters the SSB, the leading-strand DNA polymerase is able to synthesize DNA to the edge of the SSB, creating a blunt-ended recombination intermediate.     

Holocentric plant meiosis: first sisters,then homologues

Meiosis is a crucial process of sexual reproduction by forming haploid gametes from diploid precursor cells. It involves 2 subsequent divisions (meiosis I and meiosis II) after one initial round of DNA replication. Homologous monocentric chromosomes are separated during the first and sister chromatids during the second meiotic division. The faithful segregation of monocentric chromosomes is realized by mono-orientation of fused sister kinetochores at metaphase I and by bi-orientation of sister kinetochores at metaphase II. Conventionally this depends on a 2-step loss of cohesion, along chromosome arms during meiosis I and at sister centromeres during meiosis II.     

RTS1-an eukaryotic terminator of replication

Eukaryotic replication termination generally occurs randomly in the region between two active origins. However, termination, or pausing of the replication forks has been observed at specific loci. Recently, a site-specific terminator of replication named RTS1 was shown to play an important role in mating-type switching in Schizosaccharomyces pombe. Mating-type switching in S. pombe relies on an imprinting event that chemically modifies one strand of the DNA at the mating-type locus mat1. This imprint, that is formed only when mat1 is replicated in a specific direction, marks the DNA for a rearrangement leading to mating-type switching. The RTS1 element ensures that mat1 is replicated in the correct direction for imprinting and initiation of the subsequent mating-type switching event. This is the first replication terminator shown to play a role in cellular differentiation.     

Biochemical interactions between proteins and mat1 cis-acting sequences required for imprinting in fission yeast

DNA recombination required for mating type (mat1) switching in Schizosaccharomyces pombe is initiated by mat1 imprinting. The imprinting event is regulated by mat1 cis-acting elements and by several trans-acting factors, including swi1 (for switch), swi3, swi7, and sap1. swi1 and swi3 were previously shown to function in dictating unidirectional mat1 DNA replication by controlling replication fork movement around the mat1 region and, second, by pausing fork progression around the imprint site. With biochemical studies, we investigated whether the trans-acting factors function indirectly or directly by binding to the mat1 cis-acting sequences. First, we report the identification and DNA sequence of the swi3 gene. swi3 is not essential for viability, and, like the other factors, it exerts a stimulatory effect on imprinting. Second, we showed that only Swi1p and Swi3p interact to form a multiprotein complex and that complex formation did not require their binding to a DNA region defined by the smt-0 mutation. Third, we found that the Swi1p-Swi3p complex physically binds to a region around the imprint site where pausing of replication occurs. Fourth, the protein complex also interacted with the mat1-proximal polar terminator of replication (RTS1). These results suggest that the stimulatory effect of swi1 and swi3 on switching and imprinting occurs through interaction of the Swi1p-Swi3p complex with the mat1 regions.     

A matter of choice: the establishment of sister chromatid cohesion

Sister chromatid cohesion is the basis for the recognition of chromosomal DNA replication products for their bipolar segregation in mitosis. Fundamental to sister chromatid cohesion is the ring‐shaped cohesin complex, which is loaded onto chromosomes long before the initiation of DNA replication and is thought to hold replicated sister chromatids together by topological embrace. What happens to cohesin when the replication fork approaches, and how cohesin recognizes newly synthesized sister chromatids, is poorly understood. The characterization of a number of cohesion establishment factors has begun to provide hints as to the reactions involved. Cohesin is a member of the evolutionarily conserved family of Smc subunit‐based protein complexes that contribute to many aspects of chromosome biology by mediating long‐range DNA interactions. I propose that the establishment of cohesion equates to the selective stabilization of those cohesin‐mediated DNA interactions that link sister chromatids in the wake of replication forks.     

DNA replication-dependent formation of joint DNA molecules in Physarum polycephalum

Two-dimensional neutral/neutral agarose gel electrophoresis is used extensively to localize replication origins. This method resolves DNA structures containing replication forks. It also detects X-shaped recombination intermediates in meiotic cells, in the form of a typical vertical spike. Intriguingly, such a spike of joint DNA molecules is often detectable in replicating DNA from mitotic cells. Here, we used naturally synchronous DNA samples from Physarum polycephalum to demonstrate that postreplicative, DNA replication-dependent X-shaped DNA molecules are formed between sister chromatids. These molecules have physical properties reminiscent of Holliday junctions. Our results demonstrate frequent interactions between sister chromatids during a normal cell cycle and suggest a novel phase during DNA replication consisting of transient, joint DNA molecules formed on newly replicated DNA.     

GFP tagging of budding yeast chromosomes reveals that protein–protein interactions can mediate sister chromatid cohesion

Background Precise control of sister chromatid separation is essential for the accurate transmission of genetic information. Sister chromatids must remain linked to each other from the time of DNA replication until the onset of chromosome segregation, when the linkage must be promptly dissolved. Recent studies suggest that the machinery that is responsible for the destruction of mitotic cyclins also degrades proteins that play a role in maintaining sister chromatid linkage, and that this machinery is regulated by the spindle-assembly checkpoint. Studies on these problems in budding yeast are hampered by the inability to resolve its chromosomes by light or electron microscopy.Results We have developed a novel method for visualizing specific DNA sequences in fixed and living budding yeast cells. A tandem array of 256 copies of the Lac operator is integrated at the desired site in the genome and detected by the binding of a green fluorescent protein (GFP)–Lac repressor fusion expressed from the HIS3 promoter. Using this method, we show that sister chromatid segregation precedes the destruction of cyclin B. In mad or bub cells, which lack the spindle-assembly checkpoint, sister chromatid separation can occur in the absence of microtubules. The expression of a tetramerizing form of the GFP–Lac repressor, which can bind Lac operators on two different DNA molecules, can hold sister chromatids together under conditions in which they would normally separate.Conclusions We conclude that sister chromatid separation in budding yeast can occur in the absence of microtubule-dependent forces, and that protein complexes that can bind two different DNA molecules are capable of holding sister chromatids together.     

An analysis of the factory model for chromosome replication and segregation in bacteria

Recent advances in microscopy have given us important clues as to the nature of chromosome segregation in bacteria. Most current observations favour the view that the process is co-replicational: DNA replication forks are anchored at the cell centre, and the newly replicated DNA is moved towards the cell poles. This scheme can account for orderly segregation even at high growth rates where multiple replication cycles overlap. We argue that there are five distinct activities directly involved in co-replicational segregation dynamics. These we refer to as Push, Direct, Condense, Hold and Clear. We attempt to assign one of these roles to each protein implicated in chromosome segregation. The proposed process is very different from mitosis in eukaryotic cells and perhaps more closely resembles the formation of separate sister chromatids during DNA replication.     

Replication-related activities establish cohesion between sister chromatids

Replicated sister chromatids are held together from their synthesis in S phase to their separation in anaphase. The process of sister chromatid cohesion is essential for the proper segregation of chromosomes in eukaroytic cells. Recent studies in Saccharomyces cerevisiae have advanced our understanding of how sister chromatid cohesion is established, maintained, and dissolved during the cell cycle. Historical observations have suggested that establishment of cohesion is roughly coincident with replication fork passage. Emerging evidence now indicates that replication fork components, such as PCNA, a novel DNA polymerase, Trf4p/Pol σ (formerly Trf4p/Pol κ), and a modified clamp-loader complex, actively participate in the process of the cohesion establishment. Here, we review the molecular events in the chromosome cycle with respect to cohesion. Failure of sister chromatid cohesion results in the aneuploidy characteristic of many birth defects and tumors in humans.     

Epigenetic differences between sister chromatids?

Semi-conservative replication ensures that the DNA sequence of sister chromatids is identical except for replication errors and variation in the length of telomere repeats resulting from replicative losses and variable end processing. What happens with the various epigenetic marks during DNA replication is less clear. Many chromatin marks are likely to be copied onto both sister chromatids in conjunction with DNA replication, whereas others could be distributed randomly between sister chromatids. Epigenetic differences between sister chromatids could also emerge in a more predictable manner, for example, following processes that are associated with lagging strand DNA replication. The resulting epigenetic differences between sister chromatids could result in different gene expression patterns in daughter cells. This possibility has been difficult to test because techniques to distinguish between parental sister chromatids require analysis of single cells and are not obvious. Here, we briefly review the topic of sister chromatid epigenetics and discuss how the identification of sister chromatids in cells could change the way we think about asymmetric cell divisions and stochastic variation in gene expression between cells in general and paired daughter cells in particular.     

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.     

The mechanism of sister chromatid cohesion

Each of our cells inherit their genetic information in the form of chromosomes from a mother cell. In order that we obtain the full genetic complement, cells need to ensure that replicated chromosomes are accurately split and distributed during cell division. Mistakes in this process lead to aneuploidies, cells with supernumerous or missing chromosomes. Most aneuploid human embryos are not viable, and if they are, they develop severe birth defects. Aneuploidies later in human life are frequently found associated with the development of malignant cancer. DNA replication during S-phase is linked to segregation of the sister copies in mitosis by sister chromatid cohesion. A chromosomal protein complex, cohesin, holds replicated sister DNA strands together after their synthesis. This allows pairs of replication products to be recognised by the spindle apparatus in mitosis for segregation into opposite direction. At anaphase onset, cohesin is destroyed by a site-specific protease, separase. Here I review what we have learned about the molecular mechanism of sister chromatid cohesion. Cohesin forms a large proteinaceous ring that may hold sister chromatids by encircling and topological trapping. To understand how cohesin links newly synthesised replication products, biochemical assays to study the enzymology of cohesin will be required.     

Correct spindle elongation at the metaphase/anaphase transition is an APC-dependent event in budding yeast.

At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.     

The SUMO isopeptidase Ulp2p is required to prevent recombination-induced chromosome segregation lethality following DNA replication stress

SUMO conjugation is a key regulator of the cellular response to DNA replication stress, acting in part to control recombination at stalled DNA replication forks. Here we examine recombination-related phenotypes in yeast mutants defective for the SUMO de-conjugating/chain-editing enzyme Ulp2p. We find that spontaneous recombination is elevated in ulp2 strains and that recombination DNA repair is essential for ulp2 survival. In contrast to other SUMO pathway mutants, however, the frequency of spontaneous chromosome rearrangements is markedly reduced in ulp2 strains, and some types of rearrangements arising through recombination can apparently not be tolerated. In investigating the basis for this, we find DNA repair foci do not disassemble in ulp2 cells during recovery from the replication fork-blocking drug methyl methanesulfonate (MMS), corresponding with an accumulation of X-shaped recombination intermediates. ulp2 cells satisfy the DNA damage checkpoint during MMS recovery and commit to chromosome segregation with similar kinetics to wild-type cells. However, sister chromatids fail to disjoin, resulting in abortive chromosome segregation and cell lethality. This chromosome segregation defect can be rescued by overproducing the anti-recombinase Srs2p, indicating that recombination plays an underlying causal role in blocking chromatid separation. Overall, our results are consistent with a role for Ulp2p in preventing the formation of DNA lesions that must be repaired through recombination. At the same time, Ulp2p is also required to either suppress or resolve recombination-induced attachments between sister chromatids. These opposing defects may synergize to greatly increase the toxicity of DNA replication stress.     

Random Segregation of Chromatids at Mitosis in Saccharomyces Cerevisiae

Previous experiments suggest that mitotic chromosome segregation in some fungi is a nonrandom process in which chromatids of the same replicative age are destined for cosegregation. We have investigated the pattern of chromatid segregation in Saccharomyces cerevisiae by labeling the DNA of a strain auxotrophic for thymidine with 5-bromodeoxyuridine. The fate of DNA strands was followed qualitatively by immunofluorescence microscopy and quantitatively by microphotometry using an anti-5-bromodeoxyuridine monoclonal antibody. Chromatids of the same replicative age were distributed randomly to daughter cells at mitosis. Quantitative measurements showed that the amount of fluorescence in the daughter nuclei derived from parents with hemilabeled chromosomes diminished in intensity by one half. The concentration of 5-bromodeoxyuridine used in the experiments had little effect on the frequency of either homologous or sister chromatid exchanges. We infer that the 5-bromodeoxyuridine was distributed randomly due to mitotic segregation of chromatids and not via sister chromatid exchanges.     

Sister chromatid interactions in bacteria revealed by a site-specific recombination assay

The process of Sister Chromosome Cohesion (SCC), which holds together sister chromatids upon replication, is essential for chromosome segregation and DNA repair in eukaryotic cells. Although cohesion at the molecular level has never been described in E. coli, previous studies have reported that sister sequences remain co-localized for a period after their replication. Here, we have developed a new genetic recombination assay that probes the ability of newly replicated chromosome loci to interact physically. We show that Sister Chromatid Interaction (SCI) occurs exclusively within a limited time frame after replication. Importantly, we could differentiate sister cohesion and co-localization since factors such as MatP and MukB that reduced the co-localization of markers had no effect on molecular cohesion. The frequency of sister chromatid interactions were modulated by the activity of Topo-IV, revealing that DNA topology modulates cohesion at the molecular scale in bacteria.     

An Eco1-independent sister chromatid cohesion establishment pathway in S. cerevisiae

Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for their alignment on the spindle apparatus and segregation in mitosis. Budding yeast cohesin first associates with chromosomes in G1. Then, during DNA replication in S-phase, the replication fork-associated acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to make cohesin’s DNA binding resistant to destabilization by the Wapl protein. Whether stabilization of cohesin molecules that happen to link sister chromatids is sufficient to build sister chromatid cohesion, or whether additional reactions are required to establish these links, is not known. In addition to Eco1, several other factors contribute to cohesion establishment, including Ctf4, Ctf18, Tof1, Csm3, Chl1 and Mrc1, but little is known about their roles. Here, we show that each of these factors facilitates cohesin acetylation. Moreover, the absence of Ctf4 and Chl1, but not of the other factors, causes a synthetic growth defect in cells lacking Eco1. Distinct from acetylation defects, sister chromatid cohesion in ctf4Δ and chl1Δ cells is not improved by removing Wapl. Unlike previously thought, we do not find evidence for a role of Ctf4 and Chl1 in Okazaki fragment processing, or of Okazaki fragment processing in sister chromatid cohesion. Thus, Ctf4 and Chl1 delineate an additional acetylation-independent pathway that might hold important clues as to the mechanism of sister chromatid cohesion establishment.     

Sister chromatid exchanges induced by light flashes to 5-bromodeoxyuridine- and 5-iododeoxyuridine substituted Chinese hamster chromosomes

Incorporation of 5-bromodeoxyuridine (BUdR) or 5-iododeoxyuridine (IUdR) into the chromosomal DNA of Chinese hamster ovary (CHO) cells during two rounds of replication causes sister chromatids to be differentiated so that they can be discriminated from one another by staining and morphology. Chromatids that contain BUdR or IUdR in both DNA strands stain lighter and are less condensed than their sister chromatids with only unifilar substitution. The halogenated pyrimidine nucleosides also induce sister chromatid exchanges that can be detected without autoradiography. The frequency of these exchanges is markedly increased by exposing the cells to light flashes.     

Chiasmata promote monopolar attachment of sister chromatids and their co-segregation toward the proper pole during meiosis I

The chiasma is a structure that forms between a pair of homologous chromosomes by crossover recombination and physically links the homologous chromosomes during meiosis. Chiasmata are essential for the attachment of the homologous chromosomes to opposite spindle poles (bipolar attachment) and their subsequent segregation to the opposite poles during meiosis I. However, the overall function of chiasmata during meiosis is not fully understood. Here, we show that chiasmata also play a crucial role in the attachment of sister chromatids to the same spindle pole and in their co-segregation during meiosis I in fission yeast. Analysis of cells lacking chiasmata and the cohesin protector Sgo1 showed that loss of chiasmata causes frequent bipolar attachment of sister chromatids during anaphase. Furthermore, high time-resolution analysis of centromere dynamics in various types of chiasmate and achiasmate cells, including those lacking the DNA replication checkpoint factor Mrc1 or the meiotic centromere protein Moa1, showed the following three outcomes: (i) during the pre-anaphase stage, the bipolar attachment of sister chromatids occurs irrespective of chiasma formation; (ii) the chiasma contributes to the elimination of the pre-anaphase bipolar attachment; and (iii) when the bipolar attachment remains during anaphase, the chiasmata generate a bias toward the proper pole during poleward chromosome pulling that results in appropriate chromosome segregation. Based on these results, we propose that chiasmata play a pivotal role in the selection of proper attachments and provide a backup mechanism that promotes correct chromosome segregation when improper attachments remain during anaphase I.     

Building and breaking bridges between sister chromatids

Eukaryotic chromosomes undergo dramatic changes and movements during mitosis. These include the individualization and compaction of the two copies of replicated chromosomes (the sister chromatids) and their subsequent segregation to the daughter cells. Two multisubunit protein complexes termed 'cohesin' and 'condensin', both composed of SMC (Structural Maintenance of Chromosomes) and kleisin subunits, have emerged as crucial players in these processes. Cohesin is required for holding sister chromatids together whereas condensin, together with topoisomerase II, has an important role in organizing individual axes of sister chromatids prior to their segregation during anaphase. SMC and kleisin complexes also regulate the compaction and segregation of bacterial nucleoids. New research suggests that these ancient regulators of chromosome structure might function as topological devices that trap chromosomal DNA between 50 nm long coiled coils.