ORC is necessary at the interphase-to-mitosis transition to recruit cdc2 kinase and disassemble RPA foci

The origin-recognition complex (ORC) has an essential role in defining DNA replication origins and in chromosome segregation. Recent studies in Drosophila orc2 mutants, and in human cells depleted of ORC2, have suggested that this factor is also implicated in mitotic chromosome assembly. We asked whether ORC was required for M phase chromosome assembly independently of its function in DNA replication. We performed depletion assays and reconstitution experiments in Xenopus egg extracts, in conditions of M phase chromosome assembly coupled or uncoupled from DNA replication. We show that, although ORC is dispensable for mitotic chromosome condensation, it is necessary at the interphase-mitosis transition for proper mitotic chromosome assembly to occur in a reaction not strictly dependent on DNA replication. This function involves the recruitment to chromatin of cdc2 kinase and the chromatin disassembly of interphasic replication protein A (RPA) foci. Furthermore, we show that mutations of RPA at the cdc2 kinase site prevents RPA dissociation from chromatin and impairs mitotic chromosome assembly without affecting DNA replication. Our results support the conclusion that in addition to its role in the assembly of prereplication complexes (pre-RCs), at the G1-S transition, ORC is also required for their disassembly at mitotic entry.     

Patterns of polytene-chromosome replication in Simulium ornatipes (Diptera: Simuliidae)

Double labelling of Simulium ornatipes polytene chromosomes with H³- and C¹⁴-thymidine shows that chromosome synthesis follows three distinct phases viz. a short phase of initiation in puffs and interbands spreading to more condensed regions; a long continuous labelling phase, then a discontinuously labelled end phase as bands complete their replication in temporal sequence. Analysis of H³ labelling patterns indicates that while heterochromatic bands replicate there is no clear correlation between heterochromatic or C-banding regions and band replication time. The major characteristic governing band replication time appears to be band size and density. However, in some bands this relationship is modified, perhaps it is suggested, by DNA organisation influencing the efficiency of replicons. The existence of great variability in homologous band replication times, even within a chromosome pair, indicates that the control of band replication is highly autonomous. It is suggested that polymorphisms at the molecular level determine this variation. Replication time of active nucleolar organisers is very long in contrast to the short replication of condensed inactive organisers. This may reflect differential polytenisation of ribosomal DNA as a result of a developmental polymorphism, or the amplification of ribosomal DNA by active nucleolar organisers.     

Multiple pathways cooperate to facilitate DNA replication fork progression through alkylated DNA

Eukaryotic genomes are especially vulnerable to DNA damage during the S phase of the cell cycle, when chromosomes must be duplicated. The stability of DNA replication forks is critical to achieve faithful chromosome replication and is severely compromised when forks encounter DNA lesions. To maintain genome integrity, replication forks need to be protected by the S-phase checkpoint and DNA insults must be repaired. Different pathways help to repair or tolerate the lesions in the DNA, but their contribution to the progression of replication forks through damaged DNA is not well known. Here we show in budding yeast that, when the DNA template is damaged with the alkylating agent methyl methanesulfonate (MMS), base excision repair, homologous recombination and DNA damage tolerance pathways, together with a functional S-phase checkpoint, are essential for the efficient progression of DNA replication forks and the maintenance of cell survival. In the absence of base excision repair, replication forks stall reversibly in cells exposed to MMS. This repair reaction is necessary to eliminate the lesions that impede fork progression and has to be coordinated with recombination and damage tolerance activities to avoid fork collapse and allow forks to resume and complete chromosome replication.     

Developmental Regulation of the Tetrahymena thermophila Origin Recognition Complex

The Tetrahymena thermophila DNA replication machinery faces unique demands due to the compartmentalization of two functionally distinct nuclei within a single cytoplasm, and complex developmental program. Here we present evidence for programmed changes in ORC and MCM abundance that are not consistent with conventional models for DNA replication. As a starting point, we show that ORC dosage is critical during the vegetative cell cycle and development. A moderate reduction in Orc1p induces genome instability in the diploid micronucleus, aberrant division of the polyploid macronucleus, and failure to generate a robust intra-S phase checkpoint response. In contrast to yeast ORC2 mutants, replication initiation is unaffected; instead, replication forks elongation is perturbed, as Mcm6p levels decline in parallel with Orc1p. Experimentally induced down-regulation of ORC and MCMs also impairs endoreplication and gene amplification, consistent with essential roles during development. Unexpectedly Orc1p and Mcm6p levels fluctuate dramatically in developing wild type conjugants, increasing for early cycles of conventional micronuclear DNA replication and macronuclear anlagen replication (endoreplication phase I, rDNA gene amplification). This increase does not reflect the DNA replication load, as much less DNA is synthesized during this developmental window compared to vegetative S phase. Furthermore, although Orc1p levels transiently increase prior to endoreplication phase II, Orc1p and Mcm6p levels decline when the replication load increases and unconventional DNA replication intermediates are produced. We propose that replication initiation is re-programmed to meet different requirements or challenges during the successive stages of Tetrahymena development.     

PR-Set7-dependent lysine methylation ensures genome replication and stability through S phase

PR-Set7/SET8 is a histone H4–lysine 20 methyltransferase required for normal cell proliferation. However, the exact functions of this enzyme remain to be determined. In this study, we show that human PR-Set7 functions during S phase to regulate cellular proliferation. PR-Set7 associates with replication foci and maintains the bulk of H4-K20 mono- and trimethylation. Consistent with a function in chromosome dynamics during S phase, inhibition of PR-Set7 methyltransferase activity by small hairpin RNA causes a replicative stress characterized by alterations in replication fork velocity and origin firing. This stress is accompanied by massive induction of DNA strand breaks followed by a robust DNA damage response. The DNA damage response includes the activation of ataxia telangiectasia mutated and ataxia telangiectasia related kinase–mediated pathways, which, in turn, leads to p53-mediated growth arrest to avoid aberrant chromosome behavior after improper DNA replication. Collectively, these data indicate that PR-Set7–dependent lysine methylation during S phase is an essential posttranslational mechanism that ensures genome replication and stability.     

The Caulobacter crescentus chromosome replication origin evolved two classes of weak DnaA binding sites

The Caulobacter crescentus replication initiator DnaA and essential response regulator CtrA compete to control chromosome replication. The C. crescentus replication origin (Cori) contains five strong CtrA binding sites but only two apparent DnaA boxes, termed G-boxes (with a conserved second position G, TGATCCACA). Since clusters of DnaA boxes typify bacterial replication origins, this discrepancy suggested that C. crescentus DnaA recognizes different DNA sequences or compensates with novel DNA-binding proteins. We searched for novel DNA sites by scanning mutagenesis of the most conserved Cori DNA. Autonomous replication assays showed that G-boxes and novel W-boxes (TCCCCA) are essential for replication. Further analyses showed that C. crescentus DnaA binds G-boxes with moderate and W-boxes with very weak affinities significantly below DnaA's capacity for high-affinity Escherichia coli-boxes (TTATCCACA). Cori has five conserved W-boxes. Increasing W-box affinities increases or decreases autonomous replication depending on their strategic positions between the G-boxes. In vitro, CtrA binding displaces DnaA from proximal G-boxes and from distal W-boxes implying CtrA-DnaA competition and DnaA-DnaA cooperation between G-boxes and W-boxes. Similarly, during cell cycle progression, CtrA proteolysis coincides with DnaA binding to Cori. We also observe highly conserved W-boxes in other replication origins lacking E. coli-boxes. Therefore, strategically weak DnaA binding can be a general means of replication control.     

Differential regulation of homologous recombination at DNA breaks and replication forks by the Mrc1 branch of the S‐phase checkpoint

The Rad52 pathway has a central function in the recombinational repair of chromosome breaks and in the recovery from replication stress. Tolerance to replication stress also depends on the Mec1 kinase, which activates the DNA replication checkpoint in an Mrc1‐dependent manner in response to fork arrest. Although the Mec1 and Rad52 pathways are initiated by the same single‐strand DNA (ssDNA) intermediate, their interplay at stalled forks remains largely unexplored. Here, we show that the replication checkpoint suppresses the formation of Rad52 foci in an Mrc1‐dependent manner and prevents homologous recombination (HR) at chromosome breaks induced by the HO endonuclease. This repression operates at least in part by impeding resection of DNA ends, which is essential to generate 3′ ssDNA tails, the primary substrate of HR. Interestingly, we also observed that the Mec1 pathway does not prevent recombination at stalled forks, presumably because they already contain ssDNA. Taken together, these data indicate that the DNA replication checkpoint suppresses genomic instability in S phase by blocking recombination at chromosome breaks and permitting helpful recombination at stalled forks.     

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.     

Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Endoreplication Cycles

Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM–chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM–chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM–chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.     

Delineation of DNA replication time zones by fluorescence in situ hybridization.

Fluorescence in situ hybridization has been used to visualize specific genomic DNA sequences in interphase nuclei. In normal diploid cells, unreplicated DNA segments give singlet hybridization signals while replicated loci are characterized by doublets. The distribution of these two patterns in unsynchronized cell populations can be used to determine the S phase replication time of any DNA sequence. The validity of this approach was established by analyzing genes whose replication profiles in expressing and non-expressing cells had been determined previously by conventional methods. Using this technique it has been possible to map the replication timing topography of the DNA within and flanking the cystic fibrosis (CF) gene locus on chromosome 7. The gene itself is located within a defined time zone which is approximately 500 kb in length and is under developmental control. It is early replicating in cells which express CF but late replicating in other cell types. These time zones probably represent basic units of chromosome structure.     

The role of Cdc6 in ensuring complete genome licensing and S phase checkpoint activation

Before S phase, cells license replication origins for initiation by loading them with Mcm2-7 heterohexamers. This process is dependent on Cdc6, which is recruited to unlicensed origins. Using Xenopus egg extracts we show that although each origin can load many Mcm2-7 hexamers, the affinity of Cdc6 for each origins drops once it has been licensed by loading the first hexamers. This encourages the distribution of at least one Mcm2-7 hexamer to each origin, and thereby helps to ensure that all origins are licensed. Although Cdc6 is not essential for DNA replication once licensing is complete, Cdc6 regains a high affinity for origins once replication forks are initiated and Mcm2-7 has been displaced from the origin DNA. We show that the presence of Cdc6 during S phase is essential for the checkpoint kinase Chk1 to become activated in response to replication inhibition. These results show that Cdc6 plays multiple roles in ensuring precise chromosome duplication.     

Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation

Mcm10 is essential for chromosome replication in eukaryotic cells and was previously thought to link the Mcm2-7 DNA helicase at replication forks to DNA polymerase alpha. Here, we show that yeast Mcm10 interacts preferentially with the fraction of the Mcm2-7 helicase that is loaded in an inactive form at origins of DNA replication, suggesting a role for Mcm10 during the initiation of chromosome replication, but Mcm10 is not a stable component of the replisome subsequently. Studies with budding yeast and human cells indicated that Mcm10 chaperones the catalytic subunit of polymerase alpha and preserves its stability. We used a novel degron allele to inactivate Mcm10 efficiently and this blocked the initiation of chromosome replication without causing degradation of DNA polymerase alpha. Strikingly, the other essential helicase subunits Cdc45 and GINS were still recruited to Mcm2-7 when cells entered S-phase without Mcm10, but origin unwinding was blocked. These findings indicate that Mcm10 is required for a novel step during activation of the Cdc45-MCM-GINS helicase at DNA replication origins.     

Fission yeast cdc21, a member of the MCM protein family, is required for onset of S phase and is located in the nucleus throughout the cell cycle.

The fission yeast cdc21 protein belongs to the MCM family, implicated in the once per cell cycle regulation of chromosome replication. In budding yeast, proteins in this family are eliminated from the nucleus during S phase, which has led to the suggestion that they may serve to distinguish unreplicated from replicated DNA, as in the licensing factor model. We show here that, in contrast to the situation in budding yeast, cdc21 remains in the nucleus after S phase, as is found for related proteins in mammalian cells. We suggest that regulation of nuclear import of these proteins may not be an essential aspect of their function in chromosome replication. To determine the function of cdc21+, we have analysed the phenotype of a gene deletion. cdc21+ is required for entry into S phase and, unexpectedly, a proportion of cells depleted of the gene product are able to enter mitosis in the absence of DNA replication. These results are consistent with the view that individual proteins in the MCM family are required for all initiation events, and defective initiation may impair the coordination between mitosis and S phase.     

Changing roles of aurora-B kinase in two life cycle stages of Trypanosoma brucei

Aurora-B kinase is a chromosomal passenger protein essential for chromosome segregation and cytokinesis. In the procyclic form of Trypanosoma brucei, depletion of an aurora-B kinase homologue TbAUK1 inhibited spindle formation, mitosis, cytokinesis, and organelle replication without altering cell morphology. In the present study, an RNA interference knockdown of TbAUK1 or overexpression of inactive mutant TbAUK1-K58R in the bloodstream form also resulted in defects in spindle formation, chromosome segregation, and cytokinesis but allowed multiple rounds of nuclear DNA synthesis, nucleolus multiplication, and continuous replication of kinetoplast, basal body, and flagellum. The typical trypanosome morphology was lost to an enlarged round shape filled with microtubules. It is thus apparent that there are distinctive mechanisms of action of TbAUK1 in regulating cell division between the two developmental stages of trypanosome. While it exerts a tight control on mitosis, organelle replication, and cytokinesis in the procyclic form, it regulates cytokinesis without rigid control over either nuclear DNA synthesis or organelle replication in the bloodstream form. The molecular basis underlining these discrepancies remains to be explored.     

Dual role of the Cdc7-regulatory protein Dbf4 during yeast meiosis

The Dbf4-dependent Cdc7 kinase (DDK) is essential for chromosome duplication in all eukaryotes, but was proposed to be dispensable for yeast pre-meiotic DNA replication. This discrepancy led us to investigate the role of the unstable Cdc7-regulatory protein Dbf4 in meiosis. We show that, when Dbf4 is depleted at the time of meiotic induction, cells enter the meiotic program but do not replicate their chromosomes. Surprisingly when Dbf4 is depleted after the initiation of DNA synthesis, S phase goes to completion, but most cells arrest before anaphase I. Deletion of the cohesin Rec8 suppresses this phenotype, suggesting a distinct role of DDK for meiotic chromosome segregation. As after Cdc5 depletion, a fraction of cells undergo a single equational division suggesting a failure to mono-orient sister kinetochores. Our results demonstrate that Dbf4 is essential for DNA replication during meiosis like in vegetative cells and provide evidence for an additional role in setting up the reductional division of meiosis I.     

Genome Rearrangements Caused by Depletion of Essential DNA Replication Proteins in Saccharomyces cerevisiae

Genetic screens of the collection of ～4500 deletion mutants in Saccharomyces cerevisiae have identified the cohort of nonessential genes that promote maintenance of genome integrity. Here we probe the role of essential genes needed for genome stability. To this end, we screened 217 tetracycline-regulated promoter alleles of essential genes and identified 47 genes whose depletion results in spontaneous DNA damage. We further showed that 92 of these 217 essential genes have a role in suppressing chromosome rearrangements. We identified a core set of 15 genes involved in DNA replication that are critical in preventing both spontaneous DNA damage and genome rearrangements. Mapping, classification, and analysis of rearrangement breakpoints indicated that yeast fragile sites, Ty retrotransposons, tRNA genes, early origins of replication, and replication termination sites are common features at breakpoints when essential replication genes that suppress chromosome rearrangements are downregulated. We propose mechanisms by which depletion of essential replication proteins can lead to double-stranded DNA breaks near these features, which are subsequently repaired by homologous recombination at repeated elements.