The positioning and dynamics of origins of replication in the budding yeast nucleus

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)-tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.     

Replication dynamics in fission and budding yeasts through DNA polymerase tracking

The dynamics of eukaryotic DNA polymerases has been difficult to establish because of the difficulty of tracking them along the chromosomes during DNA replication. Recent work has addressed this problem in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae through the engineering of replicative polymerases to render them prone to incorporating ribonucleotides at high rates. Their use as tracers of the passage of each polymerase has provided a picture of unprecedented resolution of the organization of replicons and replication origins in the two yeasts and has uncovered important differences between them. Additional studies have found an overlapping distribution of DNA polymorphisms and the junctions of Okazaki fragments along mononucleosomal DNA. This sequence instability is caused by the premature release of polymerase δ and the retention of non proof‐read DNA tracts replicated by polymerase α. The possible implementation of these new experimental approaches in multicellular organisms opens the door to the analysis of replication dynamics under a broad range of genetic backgrounds and physiological or pathological conditions.     

Metazoan origins of DNA replication: Regulation through dynamic chromatin structure

DNA replication in eukaryotes is initiated at multiple replication origins distributed over the entire genome, which are normally activated once per cell cycle. Due to the complexity of the metazoan genome, the study of metazoan replication origins and their activity profiles has been less advanced than in simpler genome systems. DNA replication in eukaryotes involves many protein–protein and protein–DNA interactions, occurring in multiple stages. As in prokaryotes, control over the timing and frequency of initiation is exerted at the initiation site. A prerequisite for understanding the regulatory mechanisms of eukaryotic DNA replication is the identification and characterization of the cis‐acting sequences that serve as replication origins and the trans‐acting factors (proteins) that interact with them. Furthermore, in order to understand how DNA replication may become deregulated in malignant cells, the distinguishing features between normal and malignant origins of DNA replication as well as the proteins that interact with them must be determined. Based on advances that were made using simple genome model systems, several proteins involved in DNA replication have been identified. This review summarizes the current findings about metazoan origins of DNA replication and their interacting proteins as well as the role of chromatin structure in their regulation. Furthermore, progress in origin identification and isolation procedures as well as potential mechanisms to inhibit their activation in cancer development and progression are discussed. J. Cell. Biochem. 106: 512–520, 2009. © 2009 Wiley‐Liss, Inc.     

Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks

Despite its evolutionarily conserved function in controlling DNA replication, the chromosomal binding sites of the budding yeast Rif1 protein are not well understood. Here, we analyse genome‐wide binding of budding yeast Rif1 by chromatin immunoprecipitation, during G1 phase and in S phase with replication progressing normally or blocked by hydroxyurea. Rif1 associates strongly with telomeres through interaction with Rap1. By comparing genomic binding of wild‐type Rif1 and truncated Rif1 lacking the Rap1‐interaction domain, we identify hundreds of Rap1‐dependent and Rap1‐independent chromosome interaction sites. Rif1 binds to centromeres, highly transcribed genes and replication origins in a Rap1‐independent manner, associating with both early and late‐initiating origins. Interestingly, Rif1 also binds around activated origins when replication progression is blocked by hydroxyurea, suggesting association with blocked forks. Using nascent DNA labelling and DNA combing techniques, we find that in cells treated with hydroxyurea, yeast Rif1 stabilises recently synthesised DNA. Our results indicate that, in addition to controlling DNA replication initiation, budding yeast Rif1 plays an ongoing role after initiation and controls events at blocked replication forks.     

Origin plasticity during budding yeast DNA replication in vitro

The separation of DNA replication origin licensing and activation in the cell cycle is essential for genome stability across generations in eukaryotic cells. Pre‐replicative complexes (pre‐RCs) license origins by loading Mcm2‐7 complexes in inactive form around DNA. During origin firing in S phase, replisomes assemble around the activated Mcm2‐7 DNA helicase. Budding yeast pre‐RCs have previously been reconstituted in vitro with purified proteins. Here, we show that reconstituted pre‐RCs support replication of plasmid DNA in yeast cell extracts in a reaction that exhibits hallmarks of cellular replication initiation. Plasmid replication in vitro results in the generation of covalently closed circular daughter molecules, indicating that the system recapitulates the initiation, elongation, and termination stages of DNA replication. Unexpectedly, yeast origin DNA is not strictly required for DNA replication in vitro, as heterologous DNA sequences could support replication of plasmid molecules. Our findings support the notion that epigenetic mechanisms are important for determining replication origin sites in budding yeast, highlighting mechanistic principles of replication origin specification that are common among eukaryotes.     

Dynamics of DNA replication factories in living cells

DNA replication occurs in microscopically visible complexes at discrete sites (replication foci) in the nucleus. These foci consist of DNA associated with replication machineries, i.e., large protein complexes involved in DNA replication. To study the dynamics of these nuclear replication foci in living cells, we fused proliferating cell nuclear antigen (PCNA), a central component of the replication machinery, with the green fluorescent protein (GFP). Imaging of stable cell lines expressing low levels of GFP-PCNA showed that replication foci are heterogeneous in size and lifetime. Time-lapse studies revealed that replication foci clearly differ from nuclear speckles and coiled bodies as they neither show directional movements, nor do they seem to merge or divide. These four dimensional analyses suggested that replication factories are stably anchored in the nucleus and that changes in the pattern occur through gradual, coordinated, but asynchronous, assembly and disassembly throughout S phase.     

Quality control in the initiation of eukaryotic DNA replication

Origins of DNA replication must be regulated to ensure that the entire genome is replicated precisely once in each cell cycle. In human cells, this requires that tens of thousands of replication origins are activated exactly once per cell cycle. Failure to do so can lead to cell death or genome rearrangements such as those associated with cancer. Systems ensuring efficient initiation of replication, while also providing a robust block to re-initiation, play a crucial role in genome stability. In this review, I will discuss some of the strategies used by cells to ensure once per cell cycle replication and provide a quantitative framework to evaluate the relative importance and efficiency of individual pathways involved in this regulation.     

Organization of sister origins and replisomes during multifork DNA replication in Escherichia coli

The replication period of Escherichia coli cells grown in rich medium lasts longer than one generation. Initiation thus occurs in the 'mother-' or 'grandmother generation'. Sister origins in such cells were found to be colocalized for an entire generation or more, whereas sister origins in slow-growing cells were colocalized for about 0.1-0.2 generations. The role of origin inactivation (sequestration) by the SeqA protein in origin colocalization was studied by comparing sequestration-deficient mutants with wild-type cells. Cells with mutant, non-sequesterable origins showed wild-type colocalization of sister origins. In contrast, cells unable to sequester new origins due to loss of SeqA, showed aberrant localization of origins indicating a lack of organization of new origins. In these cells, aberrant replisome organization was also found. These results suggest that correct organization of sister origins and sister replisomes is dependent on the binding of SeqA protein to newly formed DNA at the replication forks, but independent of origin sequestration. In agreement, in vitro experiments indicate that SeqA is capable of pairing newly replicated DNA molecules.     

Cdk1 and Cdk2 activity levels determine the efficiency of replication origin firing in Xenopus

In this paper, we describe how, in a model embryonic system, cyclin-dependent kinase (Cdk) activity controls the efficiency of DNA replication by determining the frequency of origin activation. Using independent approaches of protein depletion and selective chemical inhibition of a single Cdk, we find that both Cdk1 and Cdk2 are necessary for efficient DNA replication in Xenopus egg extracts. Eliminating Cdk1, Cdk2 or their associated cyclins changes replication origin spacing, mainly by decreasing frequency of activation of origin clusters. Although there is no absolute requirement for a specific Cdk or cyclin, Cdk2 and cyclin E contribute more to origin cluster efficiency than Cdk1 and cyclin A. Relative Cdk activity required for DNA replication is very low, and even when both Cdk1 and Cdk2 are strongly inhibited, some origins are activated. However, at low levels, Cdk activity is limiting for the pre-replication complex to pre-initiation complex transition, origin activation and replication efficiency. As such, unlike mitosis, initiation of DNA replication responds progressively to changes in Cdk activity at low activity levels.     

Regulation of eukaryotic DNA replication and nuclearstructure

In eukaryote,nuclear structure is a key component for the functions of eukaryotic cells.More and more evidences show that the nuclear structure plays important role in regulating DNA replication.The nuclear structure provides a physical barrier for the replication licensing,participates in the decision where DNA replication initiates,and organizes replication proteins as replication factory for DNA replication.Through these works,new concepts on the regulation of DNA replication have emerged,which will be discussed in this minireview.     

Interaction of replication factor Sld3 and histone acetyl transferase Esa1 alleviates gene silencing and promotes the activation of late and dormant replication origins

DNA replication in eukaryotes is a multi-step process that consists of three main reactions: helicase loading (licensing), helicase activation (firing), and nascent DNA synthesis (elongation). Although the contributions of some chromatin regulatory factors in the licensing and elongation reaction have been determined, their functions in the firing reaction remain elusive. In the budding yeast Saccharomyces cerevisiae, Sld3, Sld7, and Cdc45 (3–7–45) are rate-limiting in the firing reaction and simultaneous overexpression of 3–7–45 causes untimely activation of late and dormant replication origins. Here, we found that 3–7–45 overexpression not only activated dormant origins in the silenced locus, HMLα, but also exerted an anti-silencing effect at this locus. For these, interaction between Sld3 and Esa1, a conserved histone acetyltransferase, was responsible. Moreover, the Sld3–Esa1 interaction was required for the untimely activation of late origins. These results reveal the Sld3–Esa1 interaction as a novel level of regulation in the firing reaction.     

Dephosphorylation of the pre-initiation complex is critical for origin firing

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Physical mapping of origins of replication in the fission yeast Schizosaccharomyces pombe.

We isolated four fragments from the Schizosaccharomyces pombe genome that mediate autonomous replication. A two-dimensional gel analysis revealed that in each case initiation could be mapped to within the S. pombe sequences. In three of the fragments, initiation could be mapped to one discrete location. In the fourth fragment, subcloning and two-dimensional gel analysis suggested that two discrete origins of replication were located within 3 kb of each other. When in proximity, usually only one of these origins fired, suggesting origin interference. Two-dimensional gel analysis of the four origin fragments at their genomic locations demonstrated that each is used in the chromosomes, but in only a subset of cells or cell divisions. The S. pombe genome appears to contain many discrete origins, not all of which fire in any given cell and some of which are closely spaced. Not I/Sfi I mapping of the five origins from this and a previous study indicates that they are randomly distributed throughout the genome and appear to be representative of chromosomal origins of replication in this organism. We compare the features of S. pombe replication origins with those of S. cerevisiae and animal cells.     

DNA damage-induced replication arrest in Xenopus egg extracts

Chromosomal replication is sensitive to the presence of DNA-damaging alkylating agents, such as methyl methanesulfonate (MMS). MMS is known to inhibit replication though activation of the DNA damage checkpoint and through checkpoint-independent slowing of replication fork progression. Using Xenopus egg extracts, we now report an additional pathway that is stimulated by MMS-induced damage. We show that, upon incubation in egg extracts, MMS-treated DNA activates a diffusible inhibitor that blocks, in trans, chromosomal replication. The downstream effect of the inhibitor is a failure to recruit proliferating cell nuclear antigen, but not DNA polymerase alpha, to the nascent replication fork. Thus, alkylation damage activates an inhibitor that intercepts the replication pathway at a point between the polymerase alpha and proliferating cell nuclear antigen execution steps. We also show that activation of the inhibitor does not require the DNA damage checkpoint; rather, stimulation of the pathway described here results in checkpoint activation. These data describe a novel replication arrest pathway, and they also provide an example of how subpathways within the DNA damage response network are integrated to promote efficient cell cycle arrest in response to damaged DNA.