Clusters,factories and domains

During S-phase of the cell cycle, chromosomal DNA is replicated according to a complex replication timing program, with megabase-sized domains replicating at different times. DNA fibre analysis reveals that clusters of adjacent replication origins fire near-synchronously. Analysis of replicating cells by light microscopy shows that DNA synthesis occurs in discrete foci or factories. The relationship between timing domains, origin clusters and replication foci is currently unclear. Recent work, using a hybrid Xenopus/hamster replication system, has shown that when CDK levels are manipulated during S-phase the activation of replication factories can be uncoupled from progression through the replication timing program. Here, we use data from this hybrid system to investigate potential relationships between timing domains, origin clusters and replication foci. We suggest that each timing domain typically comprises several replicon clusters, which are usually processed sequentially by replication factories. We discuss how replication might be regulated at different levels to create this complex organisation and the potential involvement of CDKs in this process.     

Replication timing of DNA sequences associated with human centromeres and telomeres.

The timing of replication of centromere-associated human alpha satellite DNA from chromosomes X, 17, and 7 as well as of human telomeric sequences was determined by using density-labeling methods and fluorescence-activated cell sorting. Alpha satellite sequences replicated late in S phase; however, the alpha satellite sequences of the three chromosomes studied replicated at slightly different times. Human telomeres were found to replicate throughout most of S phase. These results are consistent with a model in which multiple initiations of replication occur at a characteristic time within the alpha satellite repeats of a particular chromosome, while the replication timing of telomeric sequences is determined by either telomeric origins that can initiate at different times during S phase or by replication origins within the flanking chromosomal DNA sequences.     

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.     

Coordination between chromosome replication, segregation, and cell division in Caulobacter crescentus

Progression through the Caulobacter crescentus cell cycle is coupled to a cellular differentiation program. The swarmer cell is replicationally quiescent, and DNA replication initiates at the swarmer-to-stalked cell transition. There is a very short delay between initiation of DNA replication and movement of one of the newly replicated origins to the opposite pole of the cell, indicating the absence of cohesion between the newly replicated origin-proximal parts of the Caulobacter chromosome. The terminus region of the chromosome becomes located at the invaginating septum in predivisional cells, and the completely replicated terminus regions stay associated with each other after chromosome replication is completed, disassociating very late in the cell cycle shortly before the final cell division event. Invagination of the cytoplasmic membrane occurs earlier than separation of the replicated terminus regions and formation of separate nucleoids, which results in trapping of a chromosome on either side of the cell division septum, indicating that there is not a nucleoid exclusion phenotype.     

Replication timing in a single human chromosome 11 transferred into the Chinese hamster ovary (CHO) cell line

DNA replication in eukaryotes initiates from discrete genomic regions, termed origins, according to a strict and often tissue-specific temporal program. However, the genetic program that controls activation of replication origins has still not been fully elucidated in mammalian cells. Previously, we measured replication timing at the sequence level along human chromosomes 11q and 21q. In the present study, we sought to obtain a greater understanding of the relationship between replication timing programs and human chromosomes by analysis of the timing of replication of a single human chromosome 11 that had been transferred into the Chinese hamster ovary (CHO) cell line by chromosome engineering. Timing of replication was compared for three 11q chromosomal regions in the transformed CHO cell line (CHO(h11)) and the original human fibroblast cell line, namely, the R/G-band boundary at 11q13.5/q14.1, the centromere and the distal telomere. We found that the pattern of replication timing in and around the R/G band boundary at 11q13.5/q14.1 was similar in CHO(h11) cells and fibroblasts. The 11q centromeric region, which replicates late in human fibroblasts, replicated in the second half of S phase in CHO(h11) cells. By contrast, however, the telomeric region at 11q25, which is late replicating in fibroblasts (and in several other human cell lines), replicated in the first half of S phase or in very early S phase in CHO(h11) cells. Our observations suggest that the replication timing programs of the R/G-band boundary and the centromeric region of human chromosome 11q are maintained in CHO(h11) cells, whereas that for the telomeric region is altered. The replication timing program of telomeric regions on human chromosomes might be regulated by specific mechanisms that differ from those for other chromosomal regions.     

Temporal order of replication of mouse ribosomal RNA genes during the cell cycle

The timing of replication of mouse ribosomal RNA (rRNA) genes was determined in cultured cells by using 5-bromodeoxyuridine labeling of DNA coupled with synchronization. Two subclasses of rRNA genes were characterized that differ in their temporal order of replication during S-phase. Approximately half of the rDNA repeat units replicated primarily during the first half of S-phase and the other 50% preferentially in the second half. This difference in replication timing was consistently observed for the approximately 400 rDNA repeat units of NIH3T3 fibroblasts, but not for plasmid DNA containing fragments of rRNA genes that had been stably transfected into the genome of these cells. The rDNA fragments inserted into these transfection vectors contained the recently mapped origin of bidirectional replication with or without amplification-promoting sequences, or none of the above. Since the plasmid DNA that was integrated into the host cell genome replicated randomly during S-phase we conclude that the integrated plasmid DNA is either replicated from a chromosomal origin in the neighborhood of its integration site or that inserts are replicated from their own origins and the timing of replication is determined by flanking sequences. Received: 7 July 1997; in revised form: 1 October 1997; Accepted: 1 October 1997     

The dynamics of genome replication using deep sequencing

Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology.     

Open chromatin structures regulate the efficiencies of pre-RC formation and replication initiation in Epstein-Barr virus

Whether or not metazoan replication initiates at random or specific but flexible sites is an unsolved question. The lack of sequence specificity in origin recognition complex (ORC) DNA binding complicates genome-scale chromatin immunoprecipitation (ChIP)-based studies. Epstein-Barr virus (EBV) persists as chromatinized minichromosomes that are replicated by the host replication machinery. We used EBV to investigate the link between zones of pre-replication complex (pre-RC) assembly, replication initiation, and micrococcal nuclease (MNase) sensitivity at different cell cycle stages in a genome-wide fashion. The dyad symmetry element (DS) of EBV's latent origin, a well-established and very efficient pre-RC assembly region, served as an internal control. We identified 64 pre-RC zones that correlate spatially with 57 short nascent strand (SNS) zones. MNase experiments revealed that pre-RC and SNS zones were linked to regions of increased MNase sensitivity, which is a marker of origin strength. Interestingly, although spatially correlated, pre-RC and SNS zones were characterized by different features. We propose that pre-RCs are formed at flexible but distinct sites, from which only a few are activated per single genome and cell cycle.     

The Escherichia coli SeqA protein

The Escherichia coli SeqA protein contributes to regulation of chromosome replication by preventing re-initiation at newly replicated origins. SeqA protein binds to new DNA which is hemimethylated at the adenine of GATC sequences. Most of the cellular SeqA is found complexed with the new DNA at the replication forks. In vitro the SeqA protein binds as a dimer to two GATC sites and is capable of forming a helical fiber of dimers through interactions of the N-terminal domain. SeqA can also bind, with less affinity, to fully methylated origins and affect timing of “primary” initiations. In addition to its roles in replication, the SeqA protein may also act in chromosome organization and gene regulation.     

Timing of initiation of chromosome replication in individual Escherichia coli cells.

The synchrony of initiation of chromosome replication at multiple origins within individual Escherichia coli cells was studied by a novel method. Initiation of replication was inhibited with rifampicin or chloramphenicol and after completion of ongoing rounds of replication the numbers of fully replicated chromosomes in individual cells were measured by flow cytometry. In rapidly growing cultures, with parallel replication of several chromosomes, cells will end up with 2n (n = 1, 2, 3) chromosomes if initiation occurs simultaneously at all origins. A culture with asynchronous initiation may in addition contain cells with irregular numbers (not equal to 2n) of chromosomes. The frequency of cells with irregular numbers of chromosomes is a measure of the degree of asynchrony of initiation. After inhibition of initiation and run-out of replication in rapidly growing B/r A and K-12 cultures, a small fraction of the cells (2-7%) contained 3, 5, 6 or 7 chromosomes. From these measurements it was calculated that initiation at four origins in a single cell occurred within a small fraction, 0.1, of the doubling time (tau). A dnaA(Ts) mutant strain grown at permissive temperature exhibited a very large fraction of cells with irregular numbers of chromosomes after drug treatment demonstrating virtually random timing of initiation. A similar pattern of chromosome number per cell was found after treatment of a recA strain.     

Cell cycle analysis of F''lac replication in Escherichia coli B/r.

The timing and control of replication of an F'lac plasmid was investigated in two substrains of Escherichia coli B/r lac/F'lac growing at a variety of rates. The cellular content of covalently closed circular F'lac deoxyribonucleic acid and the cellular mass at the time of F'lac replication both increased as a function of growth rate. The timing of plasmid replication during the division cycle was determined by measuring the inducibility of beta-galactosidase in cells of different ages in exponentially growing cultures. At all growth rates, the rate of induced beta-galactosidase synthesis increased in a step-wise fashion during the division cycle, indicating that the F'lac plasmid replicated at a discrete time in the cycle. At growth rates greater than one doubling per h, the cell age at F'lac replication was indistinguishable from the cell age at chromosomal lac+ replication in an isogenic F- parent. The ratio of plasmids to chromosomal origins decreased from about 0.7 to 0.4 between growth rates of 1.0 to 2.5 doublings per h. These observations are all consistent with replication of F'lac at about the same time in the division cycle as replication of the homologous chromosomal region at these growth rates. This similarity in timing of replication of homologous deoxyribonucleic acid regions was not evident in slower-growing cells.     

The activities of eukaryotic replication origins in chromatin

DNA replication initiates at chromosomal positions called replication origins. This review will focus on the activity, regulation and roles of replication origins in Saccharomyces cerevisiae. All eukaryotic cells, including S. cerevisiae, depend on the initiation (activity) of hundreds of replication origins during a single cell cycle for the duplication of their genomes. However, not all origins are identical. For example, there is a temporal order to origin activation with some origins firing early during the S-phase and some origins firing later. Recent studies provide evidence that posttranslational chromatin modifications, heterochromatin-binding proteins and nucleosome positioning can control the efficiency and/or timing of chromosomal origin activity in yeast. Many more origins exist than are necessary for efficient replication. The availability of excess replication origins leaves individual origins free to evolve distinct forms of regulation and/or roles in chromosomes beyond their fundamental role in DNA synthesis. We propose that some origins have acquired roles in controlling chromatin structure and/or gene expression. These roles are not linked obligatorily to replication origin activity per se, but instead exploit multi-subunit replication proteins with the potential to form context-dependent protein-protein interactions.