S Phase Assembly of Centromeric Heterochromatin and Cohesion

Accurate chromosome segregation in mitosis requires cohesion between sister centromeres mediated by heterochromatin. Although establishment of both silent heterochromatin and cohesion require passage through S phase, the mechanism was previously unknown. In our recent paper, we demonstrate that heterochromatin silencing and cohesion at the centromere rely on temporal activation of the conserved S phase protein kinase Hsk1-Dfp1. Hsk1-Dfp1 is needed for heterochromatin assembly downstream of Swi6 binding to chromatin; importantly, this activity is independent of the replication function of Hsk1-Dfp1. This defines a temporal connection between S phase, heterochromatin and cohesion that is independent of replication fork passage.     

Hsk1-Dfp1 is required for heterochromatin-mediated cohesion at centromeres

Heterochromatin performs a central role in chromosome segregation and stability by promoting cohesion at centromeres. Establishment of both heterochromatin-mediated silencing and cohesion requires passage through S phase, although the mechanism is unknown. Here we demonstrate that Schizosaccharomyces pombe Hsk1 (CDC7), a conserved Dbf4-dependent protein kinase (DDK) that regulates replication initiation, interacts with and phosphorylates the heterochromatin protein 1 (HP1) equivalent Swi6 (ref. 6). Hsk1 and its regulatory subunit Dfp1 function downstream of Swi6 localization to promote heterochromatin function and cohesion specifically at centromeres. This role for Hsk1-Dfp1 is separable from its replication initiation activity, providing a temporal link between S phase and centromere cohesion that is mediated by heterochromatin.     

The Hsk1(Cdc7) replication kinase regulates origin efficiency

Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or decreasing Hsk1-Dfp1 levels correspondingly increases or decreases origin efficiency. Furthermore, tethering Hsk1-Dfp1 near an origin increases the efficiency of that origin, suggesting that the effective local concentration of Hsk1-Dfp1 regulates origin firing. Using photobleaching, we show that Hsk1-Dfp1 is freely diffusible in the nucleus. These results support a model in which the accessibility of replication origins to Hsk1-Dfp1 regulates origin efficiency and provides a potential mechanistic link between chromatin structure and replication timing. By manipulating Hsk1-Dfp1 levels, we show that increasing or decreasing origin firing rates leads to an increase in genomic instability, demonstrating the biological importance of appropriate origin efficiency.     

Hsk1-Dfp1/Him1, the Cdc7-Dbf4 kinase in Schizosaccharomyces pombe, associates with Swi1, a component of the replication fork protection complex

The protein kinase Hsk1 is essential for DNA replication in Schizosaccharomyces pombe. It associates with Dfp1/Him1 to form an active complex equivalent to the Cdc7-Dbf4 protein kinase in Saccharomyces cerevisiae. Swi1 and Swi3 are subunits of the replication fork protection complex in S. pombe that is homologous to the Tof1-Csm3 complex in S. cerevisiae. The fork protection complex helps to preserve the integrity of stalled replication forks and is important for activation of the checkpoint protein kinase Cds1 in response to fork arrest. Here we describe physical and genetic interactions involving Swi1 and Hsk1-Dfp1/Him1. Dfp1/Him1 was identified in a yeast two-hybrid screen with Swi1. Hsk1 and Dfp1/Him1 both co-immunoprecipitate with Swi1. Swi1 is required for growth of a temperature-sensitive hsk1 (hsk1ts) mutant at its semi-permissive temperature. Hsk1ts cells accumulate Rad22 (Rad52 homologue) DNA repair foci at the permissive temperature, as previously observed in swi1 cells, indicating that abnormal single-stranded DNA regions form near the replication fork in hsk1ts cells. hsk1ts cells were also unable to properly delay S-phase progression in the presence of a DNA alkylating agent and were partially defective in mating type switching. These data suggest that Hsk1-Dfp1/Him1 and Swi1-Swi3 complexes have interrelated roles in stabilization of arrested replication forks.     

Regulation of initiation of S phase, replication checkpoint signaling, and maintenance of mitotic chromosome structures during S phase by Hsk1 kinase in the fission yeast

Hsk1, Saccharomyces cerevisiae Cdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1. Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2). Checkpoint defect in hsk1-89 is indicated by accumulation of cut cells at 30 degrees C. hsk1-89 displays synthetic lethality in combination with rad3 deletion, indicating that survival of hsk1-89 depends on Rad3-dependent checkpoint pathway. Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89. Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling. hsk1-89 displays apparent defect in mitosis at 37 degrees C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures. These phenotypes are similar to those of rad21-K1 and are significantly enhanced in a hsk1-89 rad21-K1 double mutant. Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase.     

Mcl1p is a polymerase alpha replication accessory factor important for S-phase DNA damage survival

Mcl1p is an essential fission yeast chromatin-binding protein that belongs to a family of highly conserved eukaryotic proteins important for sister chromatid cohesion. The essential function is believed to result from its role as a Pol1p (polymerase alpha) accessory protein, a conclusion based primarily on analogy to Ctf4p's interaction with Pol1p. In this study, we show that Mcl1p also binds to Pol1p with high affinity for the N terminus of Pol1p during S phase and DNA damage. Characterization of an inducible allele of mcl1+, (nmt41)mcl1-MH, shows that altered expression levels of Mcl1p lead to sensitivity to DNA-damaging agents and synthetic lethality with the replication checkpoint mutations rad3Delta, rqh1Delta, and hsk1-1312. Further, we find that the overexpression of the S-phase checkpoint kinase, Cds1, or the loss of Hsk1 kinase activity can disrupt Mcl1p's interaction with chromatin and Pol1p during replication arrest with hydroxyurea. We take these data to mean that Mcl1p is a dynamic component of the polymerase alpha complex during replication and is important for the replication stress response in fission yeast.     

Schizosaccharomyces pombe Hsk1p is a potential cds1p target required for genome integrity

The fission yeast Hsk1p kinase is an essential activator of DNA replication. Here we report the isolation and characterization of a novel mutant allele of the gene. Consistent with its role in the initiation of DNA synthesis, hsk1(ts) genetically interacts with several S-phase mutants. At the restrictive temperature, hsk1(ts) cells suffer abnormal S phase and loss of nuclear integrity and are sensitive to both DNA-damaging agents and replication arrest. Interestingly, hsk1(ts) mutants released to the restrictive temperature after early S-phase arrest in hydroxyurea (HU) are able to complete bulk DNA synthesis but they nevertheless undergo an abnormal mitosis. These findings indicate a second role for hsk1 subsequent to HU arrest. Consistent with a later S-phase role, hsk1(ts) is synthetically lethal with Deltarqh1 (RecQ helicase) or rad21ts (cohesin) mutants and suppressed by Deltacds1 (RAD53 kinase) mutants. We demonstrate that Hsk1p undergoes Cds1p-dependent phosphorylation in response to HU and that it is a direct substrate of purified Cds1p kinase in vitro. These results indicate that the Hsk1p kinase is a potential target of Cds1p regulation and that its activity is required after replication initiation for normal mitosis.     

Perturbation of HP1 localization and chromatin binding ability causes defects in sister-chromatid cohesion

Sister-chromatid cohesion, the machinery used in eukaryote organisms to prevent aneuploidy, tethers sister chromatids together after their replication in S phase until mitosis. Previous studies in fission yeast, Drosophila and mammals have demonstrated the requirement for the heterochromatin formation pathway for proper centromeric cohesion. However, the exact role of heterochromatin protein 1 (HP1) in sister-chromatid cohesion in mammals is still unknown. In this study, we disrupted endogenous HP1 expression in HeLa cells using a dominant-negative mutant of HP1beta and wild-type or mutant forms of HP1alpha. We then examined their effects on chromosome alignment, segregation and cohesion. Enforced expression of these constructs leads to frequent chromosome misalignment and missegregation. Mitotic chromosomes from these cells also exhibit a loosened primary constriction and separated sister chromatids. We further demonstrate that alignment of the cohesin proteins around kinetochores was also aberrant and that cohesin complexes bound less tightly in these cells. Unexpectedly, we observed a "wavy" chromosome morphology resembling that seen upon depletion of condensin proteins in cells with over-expression of HP1alpha, but not in cells expressing the HP1beta mutant. These results indicate that proper HP1 status is required for sister-chromatid cohesion in mammalian cells, and suggest that HP1alpha might be required for chromosome condensation.     

Centromere identity in Drosophila is not determined in vivo by replication timing

Centromeric chromatin is uniquely marked by the centromere-specific histone CENP-A. For assembly of CENP-A into nucleosomes to occur without competition from H3 deposition, it was proposed that centromeres are among the first or last sequences to be replicated. In this study, centromere replication in Drosophila was studied in cell lines and in larval tissues that contain minichromosomes that have structurally defined centromeres. Two different nucleotide incorporation methods were used to evaluate replication timing of chromatin containing CID, a Drosophila homologue of CENP-A. Centromeres in Drosophila cell lines were replicated throughout S phase but primarily in mid S phase. However, endogenous centromeres and X-derived minichromosome centromeres in vivo were replicated asynchronously in mid to late S phase. Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase. We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase. These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.     

Hsk1 kinase and Cdc45 regulate replication stress-induced checkpoint responses in fission yeast

In fission yeast, replication fork arrest activates the replication checkpoint effector kinase Cds1^Chk2/Rad53 through the Rad3^ATR/Mec1-Mrc1^Claspin pathway. Hsk1, the Cdc7 homologue of fission yeast required for efficient initiation of DNA replication, is also required for Cds1 activation. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3. Rad3 kinase activity does not change in an hsk1 temperature-sensitive mutant, and Hsk1 kinase activity is not affected by rad3 mutation. Hsk1 kinase vigorously phosphorylates Mrc1 in vitro, predominantly at non-SQ/TQ sites, but this phosphorylation does not seem to affect the Rad3 action on Mrc1. Interestingly, the replication stress-induced activation of Cds1 and hyperphosphorylation of Mrc1 is almost completely abrogated in an initiation-defective mutant of cdc45, but not in an mcm2 or polε mutant. The results suggest that Hsk1-mediated loading of Cdc45 onto replication origins may play important roles in replication stress-induced checkpoint.     

RFCCtf18 and the Swi1-Swi3 complex function in separate and redundant pathways required for the stabilization of replication forks to facilitate sister chromatid cohesion in Schizosaccharomyces pombe

Sister chromatid cohesion is established during S phase near the replication fork. However, how DNA replication is coordinated with chromosomal cohesion pathway is largely unknown. Here, we report studies of fission yeast Ctf18, a subunit of the RFC(Ctf18) replication factor C complex, and Chl1, a putative DNA helicase. We show that RFC(Ctf18) is essential in the absence of the Swi1-Swi3 replication fork protection complex required for the S phase stress response. Loss of Ctf18 leads to an increased sensitivity to S phase stressing agents, a decreased level of Cds1 kinase activity, and accumulation of DNA damage during S phase. Ctf18 associates with chromatin during S phase, and it is required for the proper resumption of replication after fork arrest. We also show that chl1Delta is synthetically lethal with ctf18Delta and that a dosage increase of chl1(+) rescues sensitivities of swi1Delta to S phase stressing agents, indicating that Chl1 is involved in the S phase stress response. Finally, we demonstrate that inactivation of Ctf18, Chl1, or Swi1-Swi3 leads to defective centromere cohesion, suggesting the role of these proteins in chromosome segregation. We propose that RFC(Ctf18) and the Swi1-Swi3 complex function in separate and redundant pathways essential for replication fork stabilization to facilitate sister chromatid cohesion in fission yeast.     

Hsk1 kinase and Cdc45 regulate replication stress-induced checkpoint responses in fission yeast

In fission yeast, replication fork arrest activates the replication checkpoint effector kinase Cds1^Chk2/Rad53 through the Rad3^ATR/Mec1-Mrc1^Claspin pathway. Hsk1, the Cdc7 homolog of fission yeast required for efficient initiation of DNA replication, is also required for Cds1 activation. Hsk1 kinase activity is required for induction and maintenance of Mrc1 hyperphosphorylation, which is induced by replication fork block and mediated by Rad3. Rad3 kinase activity does not change in an hsk1 temperature-sensitive mutant, and Hsk1 kinase activity is not affected by rad3 mutation. Hsk1 kinase vigorously phosphorylates Mrc1 in vitro, predominantly at non-SQ/TQ sites, but this phosphorylation does not seem to affect the Rad3 action on Mrc1. Interestingly, the replication stress-induced activation of Cds1 and hyperphosphorylation of Mrc1 is almost completely abrogated in an initiation-defective mutant of cdc45, but not significantly in an mcm2 or polε mutant. These results suggest that Hsk1-mediated loading of Cdc45 onto replication origins may play important roles in replication stress-induced checkpoint.Key words: Cdc7, Cdc45, checkpoint, DNA replication, Mrc1     

S-phase cyclin-dependent kinases promote sister chromatid cohesion in budding yeast

Genome stability depends on faithful chromosome segregation, which relies on maintenance of chromatid cohesion during S phase. In eukaryotes, Pds1/securin is the only known inhibitor that can prevent loss of cohesion. However, pds1Δ yeast cells and securin-null mice are viable. We sought to identify redundant mechanisms that promote cohesion within S phase in the absence of Pds1 and found that cells lacking the S-phase cyclins Clb5 and Clb6 have a cohesion defect under conditions of replication stress. Similar to the phenotype of pds1Δ cells, loss of cohesion in cells lacking Clb5 and Clb6 is dependent on Esp1. However, Pds1 phosphorylation by Cdk-cyclin is not required for cohesion. Moreover, cells lacking Clb5, Clb6, and Pds1 are inviable and lose cohesion during an unperturbed S phase, indicating that Pds1 and specific B-type cyclins promote cohesion independently of one another. Consistent with this, we find that Mcd1/Scc1 is less abundant on chromosomes in cells lacking Clb5 and Clb6 during replication stress. However, clb5Δ clb6Δ cells do accumulate Mcd1/Scc1 at centromeres upon mitotic arrest, suggesting that the cyclin-dependent mechanism is S phase specific. These data indicate that Clb5 and Clb6 promote cohesion which is then protected by Pds1 and that both mechanisms are required during replication stress.     

Bipartite binding of a kinase activator activates Cdc7-related kinase essential for S phase

Dfp1/Him1 protein of fission yeast, Schizosaccharomyces pombe, encodes the regulatory subunit for Hsk1 kinase, a homologue of budding yeast Cdc7 kinase essential for initiation and progression of the S phase of the cell cycle. This protein binds and activates Hsk1 kinase, which phosphorylates the MCM2 protein. Comparison of the amino acid sequences of the Cdc7 regulatory subunits from various eukaryotes revealed the presence of three small stretches of conserved amino acid sequences, namely Dbf4 motifs N, M, and C. We report here that the Dbf4 motif M, a unique proline-rich motif, and the Dbf4 motif C, a C(2)H(2)-type zinc finger motif, are essential for mitotic functions of Dfp1/Him1 protein as well as for full-level activation of Hsk1 kinase. In vitro, a small segment containing the Dbf4 motif M or C alone binds to and partially activates Hsk1. Co-expression of these two segments augments the extent of activation. Furthermore, a fused polypeptide containing only Dbf4 motifs M and C without any spacer can activate Hsk1 and is capable of rescuing the growth defect of him1 null cells. Insertion of a long stretch of amino acids between the motif M and motif C can be tolerated for mitotic functions. On the other hand, internal deletion of Dbf4 motif N, which has some similarity with the BRCA C-terminal domain motif, results in a defect in hydroxyurea-induced checkpoint responses and sensitivity to methyl methane sulfonate, yet mitotic functions and kinase activation are intact. In one-hybrid assays with budding yeast Dbf4, motif N mutants exhibit reduced interaction with a replication origin. Our observations suggest the molecular architecture of Cdc7.Dbf4-related kinase complexes at the origins, in which they are tethered to replication machinery through Dbf4 motif N and the catalytic subunits are activated through bipartite binding of Dbf4 motifs M and C of the regulatory subunits.     

The replication timing program of the Chinese hamster beta-globin locus is established coincident with its repositioning near peripheral heterochromatin in early G1 phase

We have examined the dynamics of nuclear repositioning and the establishment of a replication timing program for the actively transcribed dihydrofolate reductase (DHFR) locus and the silent beta-globin gene locus in Chinese hamster ovary cells. The DHFR locus was internally localized and replicated early, whereas the beta-globin locus was localized adjacent to the nuclear periphery and replicated during the middle of S phase, coincident with replication of peripheral heterochromatin. Nuclei were prepared from cells synchronized at various times during early G1 phase and stimulated to enter S phase by introduction into Xenopus egg extracts, and the timing of DHFR and beta-globin replication was evaluated in vitro. With nuclei isolated 1 h after mitosis, neither locus was preferentially replicated before the other. However, with nuclei isolated 2 or 3 h after mitosis, there was a strong preference for replication of DHFR before beta-globin. Measurements of the distance of DHFR and beta-globin to the nuclear periphery revealed that the repositioning of the beta-globin locus adjacent to peripheral heterochromatin also took place between 1 and 2 h after mitosis. These results suggest that the CHO beta-globin locus acquires the replication timing program of peripheral heterochromatin upon association with the peripheral subnuclear compartment during early G1 phase.     

Cloning and characterization of hCTF18, hCTF8, and hDCC1. Human homologs of a Saccharomyces cerevisiae complex involved in sister chromatid cohesion establishment

A growing body of evidence suggests that establishment of sister chromatid cohesion is dependent on replication fork passage over a precohesion area. In Saccharomyces cerevisiae, this process involves an alternative replication factor C (RFC) complex that contains the four small RFC subunits as well as CTF18, CTF8, and DCC1. Here, we show that an evolutionarily conserved homologous complex exists in the nucleus of human cells. We demonstrate that hCTF18, hCTF8, and hDCC1 interact with each other as well as with the p38 subunit of RFC. This alternative RFC-containing complex interacts with proliferating cell nuclear antigen but not with the Rad9/Rad1/Hus1 complex, a proliferating cell nuclear antigen-like clamp involved in the DNA damage response. hCTF18 preferentially binds chromatin during S phase, suggesting a role during replication. Our data provide evidence for the existence of an alternative RFC complex with a probable role in mammalian sister chromatid cohesion establishment.