Isochores and replication time zones: a perfect match

The mammalian genome is not a random sequence but shows a specific, evolutionarily conserved structure that becomes manifest in its isochore pattern. Isochores, i.e. stretches of DNA with a distinct sequence composition and thus a specific GC content, cause the chromosomal banding pattern. This fundamental level of genome organization is related to several functional features like the replication timing of a DNA sequence. GC richness of genomic regions generally corresponds to an early replication time during S phase. Recently, we demonstrated this interdependency on a molecular level for an abrupt transition from a GC-poor isochore to a GC-rich one in the NF1 gene region; this isochore boundary also separates late from early replicating chromatin. Now, we analyzed another genomic region containing four isochores separated by three sharp isochore transitions. Again, the GC-rich isochores were found to be replicating early, the GC-poor isochores late in S phase; one of the replication time zones was discovered to consist of one single replicon. At the boundaries between isochores, that all show no special sequence elements, the replication machinery stopped for several hours. Thus, our results emphasize the importance of isochores as functional genomic units, and of isochore transitions as genomic landmarks with a key function for chromosome organization and basic biological properties.     

Evidence that both G + C rich and G + C poor isochores are replicated early and late in the cell cycle.

Since the G + C content of a gene is correlated to that of the isochore in which it resides, and early replicating isochores are thought to be relatively G + C rich, early replicating genes should also be rich in G + C. This hypothesis is tested on a sample of 44 mammalian genes for which replication time data and sequence information are available. Early replicating genes do not appear to be more G + C rich than late replicating genes, instead there is considerable variation in the G + C content of genes replicated during both halves of S phase. These results show that both G + C rich and poor fractions of the genome are replicated early and late in the cell cycle, and suggest that isochores are not maintained by the replication of DNA sequences in compositionally biased free nucleotide pools.     

Correlation between premeiotic DNA replication and chromatin transition at yeast recombination initiation sites

The DNA double-strand breaks (DSBs) that initiate meiotic recombination in Saccharomyces cerevisiae are preceded first by DNA replication and then by a chromatin transition at DSB sites. This chromatin transition, detected as a quantitative increase in micrococcal nuclease (MNase) sensitivity, occurs specifically at DSB sites and not at other MNase-sensitive sites. Replication and DSB formation are directly linked: breaks do not form if replication is blocked, and delaying replication of a region also delays DSB formation in that region. We report here experiments that examine the relationship between replication, the DSB-specific chromatin transition and DSB formation. Deleting replication origins (and thus delaying replication) on the left arm of one of the two parental chromosomes III affects DSBs specifically on that replication-delayed arm and not those on the normally replicating arm. Thus, replication timing determines DSB timing in cis. Delaying replication on the left arm of chromosome III also delays the chromatin transition at DSB sites on that arm but not on the normally replicating right arm. Since the chromatin transition precedes DSB formation and requires the function of many genes necessary for DSB formation, these results suggest that initial events for DSB formation in chromatin are coupled with premeiotic DNA replication.     

Biological implications of isochore boundaries in the human genome

The human genome is composed of large sequence segments with fairly homogeneous GC content, namely isochores, which have been linked to many important functions; biological implications of most isochore boundaries, however, remain elusive, partly due to the difficulty in determining these boundaries at high resolution. Using the segmentation algorithm based on the quadratic divergence, we re-determined all 79 boundaries of previously identified human isochores at single-nucleotide resolution, and then compared the boundary coordinates with other genome features. We found that 55.7% of isochore boundaries coincide with termini of repeat elements; 45.6% of isochore boundaries coincide with termini of highly conserved sequences based on alignment of 17 vertebrate genomes, i.e., the highly conserved genome sequence switches to a less or non-conserved one at the isochore boundary; some isochore boundaries coincide with abrupt change of CpG island distribution (note that one boundary can associate with more than one genome feature). In addition, sequences around isochore boundaries are highly conserved. It seems reasonable to deduce that the boundaries of all the isochores studied here would be replication timing sites in the human genome. These results suggest possible key roles of the isochore boundaries and may further our understanding of the human genome organization.     

Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha

We report that a plasmid replicating in Xenopus egg extracts becomes negatively supercoiled during replication initiation. Supercoiling requires the initiation factor Cdc45, as well as the single-stranded DNA-binding protein RPA, and therefore likely represents origin unwinding. When unwinding is prevented, Cdc45 binds to chromatin whereas DNA polymerase alpha does not, indicating that Cdc45, RPA, and DNA polymerase alpha bind chromatin sequentially at the G1/S transition. Whereas the extent of origin unwinding is normally limited, it increases dramatically when DNA polymerase alpha is inhibited, indicating that the helicase that unwinds DNA during initiation can become uncoupled from the replication fork. We discuss the implications of these results for the location of replication start sites relative to the prereplication complex.     

Biological Implications of Isochore Boundaries in the Human Genome

Abstract

The human genome is composed of large sequence segments with fairly homogeneous GC content, namely isochores, which have been linked to many important functions; biological implications of most isochore boundaries, however, remain elusive, partly due to the difficulty in determining these boundaries at high resolution. Using the segmentation algorithm based on the quadratic divergence, we re-determined all 79 boundaries of previously identified human isochores at single-nucleotide resolution, and then compared the boundary coordinates with other genome features. We found that 55.7% of isochore boundaries coincide with termini of repeat elements; 45.6% of isochore boundaries coincide with termini of highly conserved sequences based on alignment of 17 vertebrate genomes, i.e., the highly conserved genome sequence switches to a less or non-conserved one at the isochore boundary; some isochore boundaries coincide with abrupt change of CpG island distribution (note that one boundary can associate with more than one genome feature). In addition, sequences around isochore boundaries are highly conserved. It seems reasonable to deduce that the boundaries of all the isochores studied here would be replication timing sites in the human genome. These results suggest possible key roles of the isochore boundaries and may further our understanding of the human genome organization.     

Chromatin maturation depends on continued DNA-replication

The structure of [3H]thymidine pulse-labeled chromatin in lymphocytes differs from that of non-replicating chromatin by several operational criteria which are related to the higher nuclease sensitivity of replicating chromatin. These structural features of replicating chromatin rapidly disappear when the [3H]thymidine pulse is followed by a chase in the presence of an excess of non-radioactive thymidine. However, when the rate of DNA replication is reduced, as in cycloheximide-treated lymphocytes, chromatin maturation is retarded. No chromatin maturation is observed when nuclei from pulse-labeled lymphocytes are incubated in vitro in the absence of DNA precursors. In contrast, when these nuclei are incubated under conditions known to be optimal for DNA replication, the structure of replicating chromatin is efficiently converted to that of 'mature', non-replicating chromatin. We conclude that the properties of nascent DNA and/or the distance from the replication fork are important factors in chromatin maturation.     

A large-tumor-antigen-specific monoclonal antibody inhibits DNA replication of simian virus 40 minichromosomes in an in vitro elongation system.

In productively infected cells, a fraction of large-tumor antigen (T antigen) is tightly bound to replicating simian virus 40 (SV40) minichromosomes and does not dissociate at salt concentrations of greater than 1 M NaCl. We present electronmicrograms demonstrating the presence of T antigen on the replicated sections of replicating SV40 minichromosomes. We also show that the fraction of tightly bound T antigen is recognized by antibodies from mouse tumor serum and, more specifically, by a particular T-antigen-specific monoclonal antibody, PAb 1630. A second T-antigen-specific monoclonal antibody, PAb 101, does not react with the T-antigen fraction remaining on replicating SV40 chromatin at high salt concentrations. We used an in vitro replication system which allows, via semiconservative DNA replication, the completion of in vivo-initiated replicative intermediate DNA molecules. We show that monoclonal antibody PAb 1630, but not monoclonal antibody PAb 101, inhibits viral DNA replication. We discuss the possibility that SV40 T antigen may play a role in chain elongation during SV40 chromatin replication.     

Discovering isochores by least-squares optimal segmentation

The isochore structure of a genome is observable by variation in the G+C (guanine and cytosine) content within and between the chromosomes. Describing the isochore structure of vertebrate genomes is a challenging task, and many computational methods have been developed and applied to it. Here we apply a well-known least-squares optimal segmentation algorithm to isochore discovery. The algorithm finds the best division of the sequence into k pieces, such that the segments are internally as homogeneous as possible. We show how this simple segmentation method can be applied to isochore discovery using as input the G+C content of sliding windows on the sequence. To evaluate the performance of this segmentation technique on isochore detection, we present results from segmenting previously studied isochore regions of the human genome. Detailed results on the MHC locus, on parts of chromosomes 21 and 22, and on a 100 Mb region from chromosome 1 are similar to previously suggested isochore structures. We also give results on segmenting all 22 autosomal human chromosomes. An advantage of this technique is that oversegmentation of G+C rich regions can generally be avoided. This is because the technique concentrates on greater global, instead of smaller local, differences in the sequence composition. The effect is further emphasized by a log-transformation of the data that lowers the high variance that is observed in G+C rich regions. We conclude that the least-squares optimal segmentation method is computationally efficient and yields results close to previous biologically motivated isochore structures.     

Hepatitis B virus replication is associated with an HBx-dependent mitochondrion-regulated increase in cytosolic calcium levels

The nonstructural hepatitis B virus (HBV) protein HBx has an important role in HBV replication and in HBV-associated liver disease. Many activities have been linked to HBx expression; however, the molecular mechanisms underlying many of these activities are unknown. One proposed HBx function is the regulation of cytosolic calcium. We analyzed calcium levels in HepG2 cells that expressed HBx or replicating HBV, and we demonstrated that HBx, expressed in the absence of other HBV proteins or in the context of HBV replication, elevates cytosolic calcium. We linked this elevation of cytosolic calcium to the association of HBx with the mitochondrial permeability transition pore.     

Cell fusion-induced quick change in replication time of the inactive mouse X chromosome: an implication for the maintenance mechanism of late replication.

It is unknown how and why the genetically inactivated mammalian X chromosome replicates late in S phase. There are also occasional inactive X chromosomes characterized by an opposite behavior replicating early in S phase. Two clonal cell lines, MTLB3 and MTLH8, isolated from a cultured murine T-cell lymphoma have an allocyclic X chromosome of the latter type. This precociously replicating X chromosome was judged to be genetically inactive as the late replicating one. Immediately after fusion with another cell line, the precociously replicating X chromosome from these cells starts to replicate late in S phase. This finding seems to suggest that late replication characterizing the inactive X chromosome is actively maintained by a trans-acting factor in female somatic cells, and that its lack entails a switch from late replication to precocious replication. It remains unknown whether this presumptive factor also modifies the autosomal replication pattern.     

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.     

Superhelix density of replicating simian virus 40 DNA molecules

Simian virus 40 replicating DNA molecules were isolated and fractionated according to the extent of replication by isopynic centrifugation in ethidium bromide-CsCl. Electron microscopic examination of the replicating molecules in the presence of ethidium bromide revealed that the sense of the superhelix in replicating molecules is the same as that of simian virus 40 DNA I. Replicating DNA molecules of differing extents of replication were also analyzed by sedimentation in varying concentrations of ethidium bromide. It was observed that the superhelix density of the unreplicated portion of replicating molecules was greater than that of DNA I and that it increased as the degree of replication increased. In contrast with the increase in superhelix density that was related to the extent of replication, all replicating molecules contained a rather constant number (2 to 5) of additional superhelical turns per molecule, irrespective of the extent of replication. This suggests that a region (or regions) of about 20 to 50 nucleotides may exist in a denatured state in replicating molecules, presumably at the replicating forks of the molecule.     

Micronuclear DNA of Oxytricha nova contains sequences with autonomously replicating activity in Saccharomyces cerevisiae.

Oxytricha nova is a hypotrichous ciliate with micronuclei and macronuclei. Micronuclei, which contain large, chromosomal-sized DNA, are genetically inert but undergo meiosis and exchange during cell mating. Macronuclei, which contain only small, gene-sized DNA molecules, provide all of the nuclear RNA needed to run the cell. After cell mating the macronucleus is derived from a micronucleus, a derivation that includes excision of the genes from chromosomes and elimination of the remaining DNA. The eliminated DNA includes all of the repetitious sequences and approximately 95% of the unique sequences. We cloned large restriction fragments from the micronucleus that confer replication ability on a replication-deficient plasmid in Saccharomyces cerevisiae. Sequences that confer replication ability are called autonomously replicating sequences. The frequency and effectiveness of autonomously replicating sequences in micronuclear DNA are similar to those reported for DNAs of other organisms introduced into yeast cells. Of the 12 micronuclear fragments with autonomously replicating sequence activity, 9 also showed homology to macronuclear DNA, indicating that they contain a macronuclear gene sequence. We conclude from this that autonomously replicating sequence activity is nonrandomly distributed throughout micronuclear DNA and is preferentially associated with those regions of micronuclear DNA that contain genes.     

Studies on early replication patterns in the marsupial Sminthopsis crassicaudata: no evidence for XY replication homologies

We present here the first detailed replication banding study of a marsupial species using the BrdU-replication technique. A comparison of the structural and replication bands of the chromosomes of Sminthopsis crassicaudata clearly demonstrates that the replication behavior is the same as the described for the chromosomes of eutherians. The early replicating segments correspond to R-bands, whereas the late-replicating regions tend to be situated within Q- and C-bands. Use of this technique clearly reveals an early and late replicating X chromosome. The very small Y chromosome can be subdivided into two replication segments, but no replication homologies can be demonstrated between the X and Y chromosomes of S. crassicaudata.     

Evidence that a single replication fork proceeds from early to late replicating domains in the IgH locus in a non-B cell line

In non-B cell lines, like the murine erythroleukemia cell line (MEL), the most distal IgH constant region gene, C alpha, replicates early in S; other heavy chain constant region genes, joining and diversity segments, and the most proximal Vh gene replicate successively later in S in a 3' to 5' direction proportional to their distance from C alpha. In MEL, replication forks detected in the IgH locus also proceed in the same 3' to 5' direction for approximately 400 kb, beginning downstream of the IgH 3' regulatory region and continuing to the D region, as well as within the Vh81X gene. Downstream of the initiation region is an early replicating domain, and upstream of Vh81X is a late replicating domain. Hence, the gradual transition between early and late replicated domains can be achieved by a single replication fork.     

Correlation of GC content with replication timing and repair mechanisms in weakly expressed E.coli genes.

Regional variations of DNA GC content are observed in species as different as S.cerevisiae and humans. In vertebrates and yeast they are correlated with replication timing; late replicating chromosomal regions are more AT-rich than early replicating regions. We show here that gene composition in E.coli also has long range variations which are similarly correlated with replication timing. We suggest that the enrichment in AT base pairs in late replicating DNA reflects differences in DNA repair modes. These sequences, which are in single copy for a greater part of the cell cycle than origin-linked genes, have less opportunity to engage in repair via homologous recombination and therefore may resort more often to translesion synthesis involving the misincorporation of adenine opposite modified nucleotides.