Control over DNA replication in time and space

DNA replication is precisely regulated in time and space, thereby safeguarding genomic integrity. In eukaryotes, replication initiates from multiple sites along the genome, termed origins of replication, and propagates bidirectionally. Dynamic origin bound complexes dictate where and when replication should initiate. During late mitosis and G1 phase, putative origins are recognized and become "licensed" through the assembly of pre-replicative complexes (pre-RCs) that include the MCM2-7 helicases. Subsequently, at the G1/S phase transition, a fraction of pre-RCs are activated giving rise to the establishment of replication forks. Origin location is influenced by chromatin and nuclear organization and origin selection exhibits stochastic features. The regulatory mechanisms that govern these cell cycle events rely on the periodic fluctuation of cyclin dependent kinase (CDK) activity through the cell cycle.     

An essential role for Orc6 in DNA replication through maintenance of pre-replicative complexes

The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G(1) phase assembly of pre-replicative complexes (pre-RC). Only the Orc1-5 subunits appear to be required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. Imaging of Orc6-YFP in live cells revealed a punctate pattern consistent with the organization of replication origins into subnuclear foci. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations for metazoans, and is not required for mitosis or cytokinesis. An essential role for Orc6 in DNA replication was identified by depleting it at specific cell cycle stages. Interestingly, Orc6 was required for entry into S phase after pre-RC formation, in contrast to previous models suggesting ORC is dispensable at this point in the cell cycle. When Orc6 was depleted in late G(1), Mcm2 and Mcm10 were displaced from chromatin, cells failed to progress through S phase, and DNA combing analysis following bromodeoxyuridine incorporation revealed that the efficiency of replication origin firing was severely compromised.     

Preventing re-replication of chromosomal DNA

To ensure its duplication, chromosomal DNA must be precisely duplicated in each cell cycle, with no sections left unreplicated, and no sections replicated more than once. Eukaryotic cells achieve this by dividing replication into two non-overlapping phases. During late mitosis and G1, replication origins are 'licensed' for replication by loading the minichromosome maintenance (Mcm) 2-7 proteins to form a pre-replicative complex. Mcm2-7 proteins are then essential for initiating and elongating replication forks during S phase. Recent data have provided biochemical and structural insight into the process of replication licensing and the mechanisms that regulate it during the cell cycle.     

ORC- and Cdc6-dependent complexes at active and inactive chromosomal replication origins in Saccharomyces cerevisiae.

We have developed a genomic footprinting protocol which allows us to examine protein-DNA interactions at single copy chromosomal origins of DNA replication in the budding yeast Saccharomyces cerevisiae. We show that active replication origins oscillate between two chromatin states during the cell cycle: an origin recognition complex (ORC)-dependent post-replicative state and a Cdc6p-dependent pre-replicative state. Furthermore, we show that both post- and pre-replicative complexes can form efficiently on closely apposed replicators. Surprisingly, ARS301 which is active as an origin on plasmids but not in its normal chromosomal location, forms ORC- and Cdc6p-dependent complexes in both its active and inactive contexts. Thus, although ORC and Cdc6p are essential for initiation, their binding is not sufficient to dictate origin use.     

Limiting replication initiation factors execute the temporal programme of origin firing in budding yeast

Eukaryotic chromosomes are replicated from multiple origins that initiate throughout the S-phase of the cell cycle. Why all origins do not fire simultaneously at the beginning of S-phase is not known, but two kinase activities, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), are continually required throughout the S-phase for all replication initiation events. Here, we show that the two CDK substrates Sld3 and Sld2 and their binding partner Dpb11, together with the DDK subunit Dbf4 are in low abundance in the budding yeast, Saccharomyces cerevisiae. Over-expression of these factors is sufficient to allow late firing origins of replication to initiate early and together with deletion of the histone deacetylase RPD3, promotes the firing of heterochromatic, dormant origins. We demonstrate that the normal programme of origin firing prevents inappropriate checkpoint activation and controls S-phase length in budding yeast. These results explain how the competition for limiting DDK kinase and CDK targets at origins regulates replication initiation kinetics during S-phase and establishes a unique system with which to investigate the biological roles of the temporal programme of origin firing.     

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

To maintain genome stability, the thousands of replication origins of mammalian genomes must only initiate replication once per cell cycle. This is achieved by a strict temporal separation of ongoing replication in S phase, and the formation of pre-replicative complexes in the preceding G1 phase, which "licenses" each origin competent for replication. The contribution of the loading factor Cdc6 to the timing of the licensing process remained however elusive due to seemingly contradictory findings concerning stabilization, degradation and nuclear export of Cdc6. Using fluorescently tagged Cdc6 (Cdc6-YFP) expressed in living cycling cells, we demonstrate here that Cdc6-YFP is stable and chromatin-associated during mitosis and G1 phase. It undergoes rapid proteasomal degradation during S phase initiation followed by active export to the cytosol during S and G2 phases. Biochemical fractionation abolishes this nuclear exclusion, causing aberrant chromatin association of Cdc6-YFP and, likely, endogenous Cdc6, too. In addition, we demonstrate association of Cdc6 with centrosomes in late G2 and during mitosis. These results show that multiple Cdc6-regulatory mechanisms coexist but are tightly controlled in a cell cycle-specific manner.     

Aphidicolin triggers a block to replication origin firing in Xenopus egg extracts

DNA replication origins are located at random with respect to DNA sequence in Xenopus early embryos and on DNA replicated in Xenopus egg extracts. We have recently shown that origins fire throughout the S phase in Xenopus egg extracts. To study the temporal regulation of origin firing, we have analyzed origin activation in sperm nuclei treated with the DNA polymerase inhibitor aphidicolin. Sperm chromatin was incubated in Xenopus egg extracts in the presence of aphidicolin and transferred to a fresh extract, and digoxigenin-dUTP and biotin-dUTP were added at various times after aphidicolin release to selectively label early and late replicating DNA. Molecular combing analysis of single DNA fibers showed that only a fraction of potential origins were able to initiate in the presence of aphidicolin. After release from aphidicolin, the remaining origins fired asynchronously throughout the S phase. Therefore, initiation during the S phase depends on the normal progression of replication forks assembled at earlier activated origins. Caffeine, an inhibitor of the checkpoint kinases ATR and ATM, did not relieve the aphidicolin-induced block to origin firing. We conclude that a caffeine-insensitive intra-S phase checkpoint regulates origin activation when DNA synthesis is inhibited in Xenopus egg extracts.     

Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block-induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.     

Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome

Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics.     

Dynamic changes in chromatin structure through post‐translational modifications of histone H3 during replication origin activation

Genome duplication relies on the timely activation of multiple replication origins throughout the genome during S phase. Each origin is marked by the assembly of a multiprotein pre‐replication complex (pre‐RC) and the recruitment of the replicative machinery, which can gain access to replication origins on the DNA through the barrier of specific chromatin structures. Inheritance of the genetic information is further accompanied by maintenance and inheritance of the epigenetic marks, which are accomplished by the activity of histone and DNA modifying enzymes traveling with the replisome. Here, we studied the changes in the chromatin structure at the loci of three replication origins, the early activated human lamin B2 (LB2) and monkey Ors8 (mOrs8) origins and the late‐activated human homologue of the latter (hOrs8), during their activation, by measuring the abundance of post‐translationally modified histone H3. The data show that dynamic changes in the levels of acetylated, methylated and phosphorylated histone H3 occur during the initiation of DNA replication at these three origin loci, which differ between early‐ and late‐firing origins as well as between human‐ and monkey‐derived cell lines. These results suggest that specific histone modifications are associated with origin firing, temporal activation and replication fork progression and underscore the importance of species specificity. J. Cell. Biochem. 108: 400–407, 2009. © 2009 Wiley‐Liss, Inc.     

Genome-wide analysis of re-replication reveals inhibitory controls that target multiple stages of replication initiation

DNA replication must be tightly controlled during each cell cycle to prevent unscheduled replication and ensure proper genome maintenance. The currently known controls that prevent re-replication act redundantly to inhibit pre-replicative complex (pre-RC) assembly outside of the G1-phase of the cell cycle. The yeast Saccharomyces cerevisiae has been a useful model organism to study how eukaryotic cells prevent replication origins from reinitiating during a single cell cycle. Using a re-replication-sensitive strain and DNA microarrays, we map sites across the S. cerevisiae genome that are re-replicated as well as sites of pre-RC formation during re-replication. Only a fraction of the genome is re-replicated by a subset of origins, some of which are capable of multiple reinitiation events. Translocation experiments demonstrate that origin-proximal sequences are sufficient to predispose an origin to re-replication. Origins that reinitiate are largely limited to those that can recruit Mcm2-7 under re-replicating conditions; however, the formation of a pre-RC is not sufficient for reinitiation. Our findings allow us to categorize origins with respect to their propensity to reinitiate and demonstrate that pre-RC formation is not the only target for the mechanisms that prevent genomic re-replication.     

Mechanisms ensuring rapid and complete DNA replication despite random initiation in Xenopus early embryos

Chromosome replication initiates without sequence specificity at average intervals of approximately 10 kb during the rapid cell cycles of early Xenopus embryos. If the distribution of origins were random, some inter-origin intervals would be too long to be fully replicated before the end of S phase. To investigate what ensures rapid completion of DNA replication, we have examined the replication intermediates of plasmids of various sizes (5.3-42.2 kbp) in Xenopus egg extracts by two-dimensional gel electrophoresis and electron microscopy. We confirm that replication initiates without sequence specificity on all plasmids. We demonstrate for the first time that multiple initiation events occur on large plasmids, but not on small (<10 kb) plasmids, at average intervals of approximately 10 kb. Origin interference may prevent multiple initiation events on small plasmids. Multiple initiation events are neither synchronous nor regularly spaced. Bubble density is higher on later than on earlier replication intermediates, showing that initiation frequency increases throughout S phase, speeding up replication of late intermediates. We suggest that potential origins are abundant and randomly distributed, but that the increase of initiation frequency during S phase, and possibly origin interference, regulate origin activation to ensure rapid completion of replication.     

Nuclear matrix support of DNA replication

In higher eukaryotic cells, DNA is tandemly arranged into 10(4) replicons that are replicated once per cell cycle during the S phase. To achieve this, DNA is organized into loops attached to the nuclear matrix. Each loop represents one individual replicon with the origin of replication localized within the loop and the ends of the replicon attached to the nuclear matrix at the bases of the loop. During late G1 phase, the replication origins are associated with the nuclear matrix and dissociated after initiation of replication in S phase. Clusters of several replicons are operated together by replication factories, assembled at the nuclear matrix. During replication, DNA of each replicon is spooled through these factories, and after completion of DNA synthesis of any cluster of replicons, the respective replication factories are dismantled and assembled at the next cluster to be replicated. Upon completion of replication of any replicon cluster, the resulting entangled loops of the newly synthesized DNA are resolved by topoisomerases present in the nuclear matrix at the sites of attachment of the loops. Thus, the nuclear matrix plays a dual role in the process of DNA replication: on one hand, it represents structural support for the replication machinery and on the other, provides key protein factors for initiation, elongation, and termination of the replication of eukaryotic DNA.     

Stable association of mitotic cyclin B/Cdc2 to replication origins prevents endoreduplication

We show that in fission yeast the mitotic B type cyclin Cdc13/Cdc2 kinase associates with replication origins in vivo. This association is dependent on the origin recognition complex (ORC), is established as chromosomes are replicated, and is maintained during G2 and early mitosis. Cells expressing an orp2 (ORC2) allele that reduces binding of Cdc13 to replication origins are acutely prone to chromosomal reduplication. In synchronized endoreduplicating cells, following Cdc13 ablation, replication origins are coordinately licensed prior to each successive round of S phase with the same periodicity as in a normal cell cycle. Thus, ORC bound mitotic Cyclin B/Cdc2 kinase imposes the dependency of S phase on an intervening mitosis but not the temporal licensing of replication origins between each S phase.     

A Xenopus Dbf4 homolog is required for Cdc7 chromatin binding and DNA replication

Background  

Early in the cell cycle a pre-replicative complex (pre-RC) is assembled at each replication origin. This process involves the sequential assembly of the Origin Recognition Complex (ORC), Cdc6, Cdt1 and the MiniChromosome Maintenance (Mcm2-7) proteins onto chromatin to license the origin for use in the subsequent S phase. Licensed origins must then be activated by S phase-inducing cyclin-dependent kinases (S-CDKs) and the Dbf4/Cdc7 kinase.     

Dynamics of association of origins of DNA replication with the nuclear matrix during the cell cycle

DNA of replication foci attached to the nuclear matrix was isolated from Chinese hamster ovary cells and human HeLa cells synchronized at different stages of the G₁ and S phases of the cell cycle. The abundance of sequences from dihydrofolate reductase ori-β and the β-globin replicator was determined in matrix-attached DNA. The results show that matrix-attached DNA isolated from cells in late G₁ phase was enriched in origin sequences in comparison with matrix-attached DNA from early G₁ phase cells. The concentration of the early firing ori-β in DNA attached to the matrix decreased in early S phase, while the late firing β-globin origin remained attached until late S phase. We conclude that replication origins associate with the nuclear matrix in late G₁ phase and dissociate after initiation of DNA replication in S phase.     

Functional interactions of DNA topoisomerases with a human replication origin

The human DNA replication origin, located in the lamin B2 gene, interacts with the DNA topoisomerases I and II in a cell cycle-modulated manner. The topoisomerases interact in vivo and in vitro with precise bonds ahead of the start sites of bidirectional replication, within the pre-replicative complex region; topoisomerase I is bound in M, early G1 and G1/S border and topoisomerase II in M and the middle of G1. The Orc2 protein competes for the same sites of the origin bound by either topoisomerase in different moments of the cell cycle; furthermore, it interacts on the DNA with topoisomerase II during the assembly of the pre-replicative complex and with DNA-bound topoisomerase I at the G1/S border. Inhibition of topoisomerase I activity abolishes origin firing. Thus, the two topoisomerases are closely associated with the replicative complexes, and DNA topology plays an essential functional role in origin activation.     

Histone acetylation regulates the time of replication origin firing

The temporal firing of replication origins throughout S phase in yeast depends on unknown determinants within the adjacent chromosomal environment. We demonstrate here that the state of histone acetylation of surrounding chromatin is an important regulator of temporal firing. Deletion of RPD3 histone deacetylase causes earlier origin firing and concurrent binding of the replication factor Cdc45p to origins. In addition, increased acetylation of histones in the vicinity of the late origin ARS1412 by recruitment of the histone acetyltransferase Gcn5p causes ARS1412 alone to fire earlier. These data indicate that histone acetylation is a direct determinant of the timing of origin firing.     

The Cdc6 protein is ubiquitinated in vivo for proteolysis in Saccharomyces cerevisiae

The Saccharomyces cerevisiae Cdc6 protein is necessary for the formation of pre-replicative complexes that are required for firing DNA replication at origins at the beginning of S phase. Cdc6p protein levels oscillate during the cell cycle. In a normal cell cycle the presence of this protein is restricted to G1, partly because the CDC6 gene is transcribed only during G1 and partly because the Cdc6p protein is rapidly degraded at late G1/early S phase. We report here that the Cdc6p protein is degraded in a Cdc4-dependent manner, suggesting that phosphorylated Cdc6 is specifically recognized by the ubiquitin-mediated proteolysis machinery. Indeed, we have found that Cdc6 is ubiquitinated in vivo and degraded by a Cdc4-dependent mechanism. Our data, together with previous observations regarding Cdc6 stability, suggest that under physiological conditions budding yeast cells degrade ubiquitinated Cdc6 every cell cycle at the beginning of S phase.