染色体结构维持蛋白Smc5/6复合体的结构与功能

真核生物基因组DNA主要以染色体的形式存在于细胞核中，染色体结构的稳定及其动态变化对于真核生物遗传信息从亲代到子代中的准确传递和维持细胞的正常功能是必不可少的。染色体结构维持蛋白（Structure Maintenance of Chromosome，Smc）在染色体结构维持及DNA损伤修复方面发挥着关键性的作用。Smc蛋白家族包括3类：黏连蛋白（Cohesin）、凝缩蛋白（Condesin）和Smc5/6复合体（Smc5/6 complex）。Smc5/6复合体在真核生物中分布广泛且高度保守，在DNA损伤同源重组修复、DNA复制、端粒长度维持及胚胎发育中发挥重要作用。本文系统介绍了Smc5/6复合体结构特征和生物学功能领域的相关进展，为深入研究Smc5/6复合体提供理论参考。     

Microtubule-motor activity of a yeast centromere-binding protein complex.

During cell division, sister chromosomes segregate from each other on a microtubule-based structure called the mitotic spindle. Proteins bind to the centromere, a region of chromosomal DNA, to form the kinetochore, which mediates chromosome attachment to the mitotic spindle microtubules. In the budding yeast Saccharomyces cerevisiae, genetic analysis has shown that the 28-basepair (bp) CDEIII region of the 125-bp centromere DNA sequence (CEN sequence) is the main region controlling chromosome segregation in vivo. Therefore it is likely that proteins binding to the CDEIII region link the centromeres to the microtubules during mitosis. A complex of proteins (CBF3) that binds specifically to the CDEIII DNA sequence has been isolated by affinity chromatography. Here we describe kinetochore function in vitro. The CBF3 complex can link DNA to microtubules, and the complex contains a minus-end-directed microtubule-based motor. We suggest that microtubule-based motors form the fundamental link between microtubules and chromosomes at mitosis.     

Aberrant chromosome morphology in human cells defective for Holliday junction resolution

In somatic cells, Holliday junctions can be formed between sister chromatids during the recombinational repair of DNA breaks or after replication fork demise. A variety of processes act upon Holliday junctions to remove them from DNA, in events that are critical for proper chromosome segregation. In human cells, the BLM protein, inactivated in individuals with Bloom's syndrome, acts in combination with topoisomerase IIIα, RMI1 and RMI2 (BTR complex) to promote the dissolution of double Holliday junctions. Cells defective for BLM exhibit elevated levels of sister chromatid exchanges (SCEs) and patients with Bloom's syndrome develop a broad spectrum of early-onset cancers caused by chromosome instability. MUS81-EME1 (refs 4-7), SLX1-SLX4 (refs 8-11) and GEN1 (refs 12, 13) also process Holliday junctions but, in contrast to the BTR complex, do so by endonucleolytic cleavage. Here we deplete these nucleases from Bloom's syndrome cells to analyse human cells compromised for the known Holliday junction dissolution/resolution pathways. We show that depletion of MUS81 and GEN1, or SLX4 and GEN1, from Bloom's syndrome cells results in severe chromosome abnormalities, such that sister chromatids remain interlinked in a side-by-side arrangement and the chromosomes are elongated and segmented. Our results indicate that normally replicating human cells require Holliday junction processing activities to prevent sister chromatid entanglements and thereby ensure accurate chromosome condensation. This phenotype was not apparent when both MUS81 and SLX4 were depleted from Bloom's syndrome cells, suggesting that GEN1 can compensate for their absence. Additionally, we show that depletion of MUS81 or SLX4 reduces the high frequency of SCEs in Bloom's syndrome cells, indicating that MUS81 and SLX4 promote SCE formation, in events that may ultimately drive the chromosome instabilities that underpin early-onset cancers associated with Bloom's syndrome.     

Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1.

Cohesion between sister chromatids is established during DNA replication and depends on a multiprotein complex called cohesin. Attachment of sister kinetochores to the mitotic spindle during mitosis generates forces that would immediately split sister chromatids were it not opposed by cohesion. Cohesion is essential for the alignment of chromosomes in metaphase but must be abolished for sister separation to start during anaphase. In the budding yeast Saccharomyces cerevisiae, loss of sister-chromatid cohesion depends on a separating protein (separin) called Esp1 and is accompanied by dissociation from the chromosomes of the cohesion subunit Scc1. Here we show that Esp1 causes the dissociation of Scc1 from chromosomes by stimulating its cleavage by proteolysis. A mutant Scc1 is described that is resistant to Esp1-dependent cleavage and which blocks both sister-chromatid separation and the dissociation of Scc1 from chromosomes. The evolutionary conservation of separins indicates that the proteolytic cleavage of cohesion proteins might be a general mechanism for triggering anaphase.     

The cohesin ring concatenates sister DNA molecules

Sister chromatid cohesion, which is essential for mitosis, is mediated by a multi-subunit protein complex called cohesin. Cohesin's Scc1, Smc1 and Smc3 subunits form a tripartite ring structure, and it has been proposed that cohesin holds sister DNA molecules together by trapping them inside its ring. To test this, we used site-specific crosslinking to create chemical connections at the three interfaces between the three constituent polypeptides of the ring, thereby creating covalently closed cohesin rings. As predicted by the ring entrapment model, this procedure produced dimeric DNA-cohesin structures that are resistant to protein denaturation. We conclude that cohesin rings concatenate individual sister minichromosome DNA molecules.     

Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA

Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The topoisomerase II mutants (top2) are conditional-lethal temperature-sensitive (ts) mutants. They are defective in the termination of DNA replication and the segregation of daughter chromosomes, but otherwise appear to replicate and transcribe DNA normally. Topoisomerase I mutants (top1), including strains with null mutations are viable and exhibit no obvious growth defects, demonstrating that DNA topoisomerase I is not essential for viability in yeast. In contrast to the single mutants, top1 top2 ts double mutants from both Schizosaccharomyces pombe and Saccharomyces cerevisiae grow poorly at the permissive temperature and stop growth rapidly at the non-permissive temperature. Here we report that DNA and ribosomal RNA synthesis are drastically inhibited in an S. cerevisiae top1 top2 ts double mutant at the restrictive temperature, but that the rate of poly(A)+ RNA synthesis is reduced only about threefold and transfer DNA synthesis remains relatively normal. The results suggest that DNA replication and at least ribosomal RNA synthesis require an active topoisomerase, presumably to act as a swivel to relieve torsional stress, and that either topoisomerase can perform the required function (except in termination of DNA replication where topoisomerase II is required).     

DNA sequences of telomeres maintained in yeast

Telomeres, the ends of eukaryotic chromosomes, have long been recognized as specialized structures. Their stability compared with broken ends of chromosomes suggested that they have properties which protect them from fusion, degradation or recombination. Furthermore, a linear DNA molecule such as that of a eukaryotic chromosome must have a structure at its ends which allows its complete replication, as no known DNA polymerase can initiate synthesis without a primer. At the ends of the relatively short, multi-copy linear DNA molecules found naturally in the nuclei of several lower eukaryotes, there are simple tandemly repeated sequences with, in the cases analysed, a specific array of single-strand breaks, on both DNA strands, in the distal portion of the block of repeats. In general, however, direct analysis of chromosomal termini presents problems because of their very low abundance in nuclei. To circumvent this problem, we have previously cloned a chromosomal telomere of the yeast Saccharomyces cerevisiae on a linear DNA vector molecule. Here we show that yeast chromosomal telomeres terminate in a DNA sequence consisting of tandem irregular repeats of the general form C1-3A. The same repeat units are added to the ends of Tetrahymena telomeres, in an apparently non-template-directed manner, during their replication on linear plasmids in yeast. Such DNA addition may have a fundamental role in telomere replication.     

Structural basis for gate-DNA recognition and bending by type IIA topoisomerases

Type II topoisomerases disentangle DNA to facilitate chromosome segregation, and represent a major class of therapeutic targets. Although these enzymes have been studied extensively, a molecular understanding of DNA binding has been lacking. Here we present the structure of a complex between the DNA-binding and cleavage core of Saccharomyces cerevisiae Topo II (also known as Top2) and a gate-DNA segment. The structure reveals that the enzyme enforces a 150 degrees DNA bend through a mechanism similar to that of remodelling proteins such as integration host factor. Large protein conformational changes accompany DNA deformation, creating a bipartite catalytic site that positions the DNA backbone near a reactive tyrosine and a coordinated magnesium ion. This configuration closely resembles the catalytic site of type IA topoisomerases, reinforcing an evolutionary link between these structurally and functionally distinct enzymes. Binding of DNA facilitates opening of an enzyme dimerization interface, providing visual evidence for a key step in DNA transport.     

A MAP kinase-dependent actin checkpoint ensures proper spindle orientation in fission yeast.

The accurate segregation of chromosomes at mitosis depends on a correctly assembled bipolar spindle that exerts balanced forces on each sister chromatid. The integrity of mitotic chromosome segregation is ensured by the spindle assembly checkpoint (SAC) that delays mitosis in response to defective spindle organisation or failure of chromosome attachment. Here we describe a distinct mitotic checkpoint in the fission yeast, Schizosaccharomyces pombe, that monitors the integrity of the actin cytoskeleton and delays sister chromatid separation, spindle elongation and cytokinesis until spindle poles have been properly oriented. This mitotic delay is imposed by a stress-activated mitogen-activated protein (MAP) kinase pathway but is independent of the anaphase-promoting complex (APC).     

S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex

The checkpoint regulatory mechanism has an important role in maintaining the integrity of the genome. This is particularly important in S phase of the cell cycle, when genomic DNA is most susceptible to various environmental hazards. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damaged DNA. Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks. However, the molecular mechanism coupling checkpoint regulation and replication arrest is unknown. Here we demonstrate that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in Saccharomyces cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing structure that serves to anchor subsequent DNA repair events.     

Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells

Double-strand breaks occur during DNA replication and are also induced by ionizing radiation. There are at least two pathways which can repair such breaks: non-homologous end joining and homologous recombination (HR). Although these pathways are essentially independent of one another, it is possible that the proteins Mre11, Rad50 and Xrs2 are involved in both pathways in Saccharomyces cerevisiae. In vertebrate cells, little is known about the exact function of the Mre11-Rad50-Nbs1 complex in the repair of double-strand breaks because Mre11- and Rad50-null mutations are lethal. Here we show that Nbs1 is essential for HR-mediated repair in higher vertebrate cells. The disruption of Nbs1 reduces gene conversion and sister chromatid exchanges, similar to other HR-deficient mutants. In fact, a site-specific double-strand break repair assay showed a notable reduction of HR events following generation of such breaks in Nbs1-disrupted cells. The rare recombinants observed in the Nbs1-disrupted cells were frequently found to have aberrant structures, which possibly arise from unusual crossover events, suggesting that the Nbs1 complex might be required to process recombination intermediates.     

ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex.

A multiprotein complex that specifically recognizes cellular origins of DNA replication has been identified and purified from the yeast Saccharomyces cerevisiae. We observe a strong correlation between origin function and origin recognition by this activity. Interestingly, specific DNA binding by the origin recognition complex is dependent upon the addition of ATP. We propose that the origin recognition complex acts as the initiator protein for S. cerevisiae origins of DNA replication.     

Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint

The checkpoint kinase proteins Mec1 and Rad53 are required in the budding yeast, Saccharomyces cerevisiae, to maintain cell viability in the presence of drugs causing damage to DNA or arrest of DNA replication forks. It is thought that they act by inhibiting cell cycle progression, allowing time for DNA repair to take place. Mec1 and Rad53 also slow S phase progression in response to DNA alkylation, although the mechanism for this and its relative importance in protecting cells from DNA damage have not been determined. Here we show that the DNA-alkylating agent methyl methanesulphonate (MMS) profoundly reduces the rate of DNA replication fork progression; however, this moderation does not require Rad53 or Mec1. The accelerated S phase in checkpoint mutants, therefore, is primarily a consequence of inappropriate initiation events. Wild-type cells ultimately complete DNA replication in the presence of MMS. In contrast, replication forks in checkpoint mutants collapse irreversibly at high rates. Moreover, the cytotoxicity of MMS in checkpoint mutants occurs specifically when cells are allowed to enter S phase with DNA damage. Thus, preventing damage-induced DNA replication fork catastrophe seems to be a primary mechanism by which checkpoints preserve viability in the face of DNA alkylation.     

Effect of novobiocin on initiation of DNA replication in Bacillus subtilis.

The initiation of DNA replication of small replicons in vitro involves conformational changes in the whole DNA molecule or in the region near to the replication origin. One striking finding has been the role of DNA gyrase (that is, the necessity for supercoiled structure) in the initial stage of ColE1 replication in vitro. However, little is known about the effect of gyrase on the initiation of replication of bacterial chromosomes in vivo. We have constructed a map of cleavage sites of restriction enzymes at the region of the origin of replication of the Bacillus subtilis chromosome (accompanying paper). This has now enabled us to examine the effect of novobiocin, a selective inhibitor of DNA gyrase, on the replication of the specific chromosomal segments near the origin and to seek a possible role for the gyrase in the initiation of chromosomal replication. We have found that only a limited segment of the chromosome at the origin region was replicated in the presence of novobiocin. This effect allowed us to locate the site of the origin of replication to within a DNA fragment of molecular weight 3.4 x 10(6).     

Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication

Cell-free replication systems for simian virus 40 (SV40) DNA are taken to be a model for the replication of eukaryotic chromosomes, because only one viral protein is required to supplement the replication proteins provided by a human cell extract. To prove that these cellular proteins function in chromosomal DNA replication we have begun to identify homologous proteins in an organism that can be genetically manipulated. Here we report the identification of yeast replication factor-A (yRF-A) from Saccharomyces cerevisiae and show that it is functionally and structurally related to a human protein that is required for the initiation and elongation of SV40 DNA replication. Yeast RF-A, a multi-subunit phosphoprotein, is similar to the human protein in its chromatographic behaviour, subunit structure and DNA-binding activity. The yeast protein will fully substitute for the human protein in an early stage of the initiation of SV40 DNA replication. Substitution of yRF-A in the complete SV40 replication system, however, results in reduced DNA replication, presumably due to a requirement for species-specific interactions between yeast RF-A and the DNA polymerase complex.     

DNA ligase I deficiency in Bloom's syndrome

Certain rare human diseases with autosomal recessive mode of inheritance are associated with a greatly increased cancer frequency which may reflect specific defects in DNA repair or replication. These disorders include xeroderma pigmentosum, ataxia-telangiectasia, Fanconi's anaemia and Bloom's syndrome. Cells from individuals with Bloom's syndrome usually grow slowly in culture and exhibit increased chromosomal breakage and rearrangement, an elevated frequency of sister chromatid exchanges, retarded rates of progression of DNA replication forks, delayed conversion of replication intermediates to high-molecular-weight DNA, and slightly increased sensitivity to DNA-damaging agents. Several of these features are also characteristic of Escherichia coli and yeast mutants with a defective DNA ligase. In this investigation we show that one of the two DNA ligases of human cells, ligase I, is defective in a representative lymphoid cell line of Bloom's syndrome origin.     

Stabilization of microtubule dynamics at anaphase onset promotes chromosome segregation

Microtubules of the mitotic spindle form the structural basis for chromosome segregation. In metaphase, microtubules show high dynamic instability, which is thought to aid the 'search and capture' of chromosomes for bipolar alignment on the spindle. Microtubules suddenly become more stable at the onset of anaphase, but how this change in microtubule behaviour is regulated and how important it is for the ensuing chromosome segregation are unknown. Here we show that in the budding yeast Saccharomyces cerevisiae, activation of the phosphatase Cdc14 at anaphase onset is both necessary and sufficient for silencing microtubule dynamics. Cdc14 is activated by separase, the protease that triggers sister chromatid separation, linking the onset of anaphase to microtubule stabilization. If sister chromatids separate in the absence of Cdc14 activity, microtubules maintain high dynamic instability; this correlates with defects in both the movement of chromosomes to the spindle poles (anaphase A) and the elongation of the anaphase spindle (anaphase B). Cdc14 promotes localization of microtubule-stabilizing proteins to the anaphase spindle, and dephosphorylation of the kinetochore component Ask1 contributes to both the silencing of microtubule turnover and successful anaphase A.     

Repetitive shuttling of a motor protein on DNA

Many helicases modulate recombination, an essential process that needs to be tightly controlled. Mutations in some human disease helicases cause increased recombination, genome instability and cancer. To elucidate the potential mode of action of these enzymes, here we developed a single-molecule fluorescence assay that can visualize DNA binding and translocation of Escherichia coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae Srs2. Individual Rep monomers were observed to move on single-stranded (ss)DNA in the 3' to 5' direction using ATP hydrolysis. Strikingly, on hitting a blockade, such as duplex DNA or streptavidin, the protein abruptly snapped back close to its initial position, followed by further cycles of translocation and snapback. This repetitive shuttling is likely to be caused by a blockade-induced protein conformational change that enhances DNA affinity for the protein's secondary DNA binding site, thereby resulting in a transient DNA loop. Repetitive shuttling was also observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment analogue, and the presence of Rep delayed formation of a filament of recombination protein RecA on ssDNA. Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates.