Replication timing of two human common fragile sites: FRA1H and FRA2G

The mammalian chromosomes present specific sites of gaps or breaks, the common fragile sites (CFSs), when the cells are exposed to DNA replication stress or to some DNA binding compounds. CFSs span hundreds or thousands of kilobases. The analysis of these sequences has not definitively clarified the causes of their fragility. There is considerable evidence that CFSs are regions of late or slowed replication in the presence of sequence elements that have the propensity to form secondary structures, and that the cytogenetic expression of CFSs may be due to unreplicated DNA. In order to analyse the relationship between DNA replication time and fragility, in this work we have investigated the timing of replication of sequences mapping within two CFSs (FRA1H and FRA2G), of syntenic non-fragile sequences and of early and late replicating control sequences by using fluorescent in situ hybridization on interphase nuclei, conventional fluorescence microscopy and confocal microscopy. Our results indicate that the fragile sequences are slow replicating and that they enter G2 phase unreplicated with very high frequency. Thus these regions could sometimes reach mitosis unreplicated or undercondensed and be expressed as chromosome gaps/breakages.     

High-resolution mapping of mitotic DNA synthesis regions and common fragile sites in the human genome through direct sequencing

DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.Subject terms: Cancer, DNA damage and repair     

A moving DNA replication factory in Caulobacter crescentus

The in vivo intracellular location of components of the Caulobacter replication apparatus was visualized during the cell cycle. Replisome assembly occurs at the chromosomal origin located at the stalked cell pole, coincident with the initiation of DNA replication. The replisome gradually moves to midcell as DNA replication proceeds and disassembles upon completion of DNA replication. Although the newly replicated origin regions of the chromosome are rapidly moved to opposite cell poles by an active process, the replisome appears to be an untethered replication factory that is passively displaced towards the center of the cell by the newly replicated DNA. These results are consistent with a model in which unreplicated DNA is pulled into the replication factory and newly replicated DNA is bidirectionally extruded from the complex, perhaps contributing to chromosome segregation.     

Tn7 transposes proximal to DNA double-strand breaks and into regions where chromosomal DNA replication terminates

We report that the bacterial transposon Tn7 can preferentially transpose into regions where chromosomal DNA replication terminates. DNA double-strand breaks are associated with the termination of chromosomal replication; therefore, we directly tested the effect of DNA breaks on Tn7 transposition. When DNA double-strand breaks are induced at specific sites in the chromosome, Tn7 transposition is stimulated and insertions are directed proximal to the induced break. The targeting preference for the terminus of replication and DNA double-strand breaks is dependent on the Tn7-encoded protein TnsE. The results presented in this study could also explain the previous observation that Tn7 is attracted to events associated with conjugal DNA replication during plasmid DNA transfer.     

Stepwise Activation of the ATR Signaling Pathway upon Increasing Replication Stress Impacts Fragile Site Integrity

Breaks at common fragile sites (CFS) are a recognized source of genome instability in pre-neoplastic lesions, but how such checkpoint-proficient cells escape surveillance and continue cycling is unknown. Here we show, in lymphocytes and fibroblasts, that moderate replication stresses like those inducing breaks at CFSs trigger chromatin loading of sensors and mediators of the ATR pathway but fail to activate Chk1 or p53. Consistently, we found that cells depleted of ATR, but not of Chk1, accumulate single-stranded DNA upon Mre11-dependent resection of collapsed forks. Partial activation of the pathway under moderate stress thus takes steps against fork disassembly but tolerates S-phase progression and mitotic onset. We show that fork protection by ATR is crucial to CFS integrity, specifically in the cell type where a given site displays paucity in backup replication origins. Tolerance to mitotic entry with under-replicated CFSs therefore results in chromosome breaks, providing a pool of cells committed to further instability.     

The nuclear location and chromatin organization of active chorion amplification origins

It remains unclear how certain regions on metazoan chromosomes are selected to initiate DNA replication. In recent years a number of origins of DNA replication have been mapped, but there is still no DNA consensus for predicting where replication will initiate. Evidence suggests that the higher order structure of the nucleus and chromosome influences origin activity. Chromosomal DNA replication is proposed to occur in special compartments in the nucleus called replication foci. Foci in different regions of the nucleus initiate replication at different times of S-phase, suggesting nuclear position may contribute to where and when replication begins. Here we test the contribution of nuclear compartments for well-defined origins, those involved in amplification of the chorion (eggshell) genes during Drosophila oogenesis. The results of three-dimensional confocal microscopy indicate that chorion DNA replication origins are highly active in diverse positions within the nucleus. We also find that chorion replication origins inserted at ectopic chromosomal sites can amplify highly in diverse nuclear locations distinct from the endogenous loci, including when they are buffered against genomic position effects. We used fluorescence in situ hybridization to analyze chromosome structure during amplification. Contrary to the replication factory model, we find no evidence for spooling of DNA toward a replication center. We discuss the implications of these results for understanding the role of higher order structure in amplification and chromosome duplication.     

High-affinity binding sites for the initiator protein DnaA on the chromosome of Escherichia coli

The initiator protein DnaA of Escherichia coli binds with unusually high affinity to five regions on the chromosome, in addition to the replication origin, oriC . Using a solid-phase DNA binding assay, in which the DNA binding C-terminal domain of DnaA is bound via a biotin tag to magnetic beads, we could fish only fragments with these six regions from different chromosomal digests. Except for oriC , these fragments contain only one or two consensus DnaA binding sites, DnaA boxes. The distribution of these high-affinity DnaA boxes on the chromosome is random.     

Mechanism of Replicative DNA Polymerase Delta Pausing and a Potential Role for DNA Polymerase Kappa in Common Fragile Site Replication

Common fragile sites (CFSs) are hot spots of chromosomal breakage, and CFS breakage models involve perturbations of DNA replication. Here, we analyzed the contribution of specific repetitive DNA sequence elements within CFSs to the inhibition of DNA synthesis by replicative and specialized DNA polymerases (Pols). The efficiency of in vitro DNA synthesis was quantitated using templates corresponding to regions within FRA16D and FRA3B harboring AT-rich microsatellite and quasi-palindrome (QP) sequences. QPs were predicted to form stems of ~ 75–100% self-homology, separated by 3–9 bases of intervening sequences. Analysis of DNA synthesis progression by human Pol δ demonstrated significant synthesis perturbation both at [A]_n and [TA]_n repeats in a length-dependent manner and at short (< 40 base pairs) QP sequences. DNA synthesis by the Y-family polymerase κ was significantly more efficient than Pol δ through both types of repetitive elements. Using DNA trap experiments, we show that Pol δ pauses within CFS sequences are sites of enzyme dissociation, and dissociation was observed in the presence of RFC-loaded PCNA. We propose that enrichment of microsatellite and QP elements at CFS regions contributes to fragility by perturbing replication through multiple mechanisms, including replicative Pol pausing and dissociation. Our finding that Pol δ dissociates at specific CFS sequences is significant, since dissociation of the replication machinery and inability to efficiently recover the replication fork can lead to fork collapse and/or formation of double-strand breaks in vivo. Our biochemical studies also extend the potential involvement of Y-family polymerases in CFS maintenance to include polymerase κ.     

Mechanisms and regulation of DNA replication initiation in eukaryotes

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms.     

Terminal regions of mammal chromosomes: plasticity and role in evolution

The review considers data on the composition, organization, and functional significance of terminal regions in mammalian chromosomes, including telomeres and subtelomeric regions. Because of specific structure, features of DNA replication, and characteristic localization in somatic and meiotic cells, these regions are hot spots for many events associated with genome functioning in mammals. Instability of these regions is of special interest. Evidence suggesting that instability of chromosomal regions containing telomeric DNA is a factor of chromosome evolution is discussed. The association of size and structure of telomeric regions with replicative aging and cell immortalization is considered. The review deals in detail with classical and alternative mechanisms of telomere size control, the significance of changes in telomeric region length in ontogeny, oncotransformation, and evolution. The issues related to telomere destabilization and the role of this process in chromosome rearrangement formation and chromosome evolution are discussed. The origin of telomere repeats in interstitial chromosome sites, including regions of evolutionary fusions-fissions is given special consideration. The possible role of ribosomal repeats and mechanisms similar to ALT (alternative lengthening of telomeres) in telomere reorganization in some taxa are discussed.     

WWOX,large common fragile site genes,and cancer

WWOX is a gene that spans an extremely large chromosomal region. It is derived from within chromosomal band 16q23.2 which is a region with frequent deletions and other alterations in a variety of different cancers. This chromosomal band also contains the FRA16D common fragile site (CFS). CFSs are chromosomal regions found in all individuals which are highly unstable. WWOX has also been demonstrated to function as a tumor suppressor that is involved in the development of many cancers. Two other highly unstable CFSs, FRA3B (3p14.2) and FRA6E (6q26), also span extremely large genes, FHIT and PARK2, respectively, and these two genes are also found to be important tumor suppressors. There are a number of interesting similarities between these three large CFS genes. In spite of the fact that they are derived from some of the most unstable chromosomal regions in the genome, they are found to be highly evolutionarily conserved and the chromosomal region spanning the mouse homologs of both WWOX and FHIT are also CFSs in mice. Many of the other CFSs also span extremely large genes and many of these are very attractive tumor suppressor candidates. WWOX is therefore a member of a very interesting family of very large CFS genes.     

Terminal regions of mammal chromosomes: Plasticity and role in evolution

The review considers data on the composition, organization, and functional significance of terminal regions in mammalian chromosomes, including telomeres and subtelomeric regions. Because of specific structure, features of DNA replication, and characteristic localization in somatic and meiotic cells, these regions are hot spots for many events associated with genome functioning in mammals. Instability of these regions is of special interest. Evidence suggesting that instability of chromosomal regions containing telomeric DNA is a factor of chromosome evolution is discussed. The association of size and structure of telomeric regions with replicative aging and cell immortalization is considered. The review deals in detail with classical and alternative mechanisms of telomere size control, the significance of changes in telomeric region length in ontogeny, oncotransformation, and evolution. The issues related to telomere destabilization and the role of this process in chromosome rearrangement formation and chromosome evolution are discussed. The origin of telomere repeats in interstitial chromosome sites, including regions of evolutionary fusions-fissions is given special consideration. The possible role of ribosomal repeats and mechanisms similar to ALT (alternative lengthening of telomeres) in telomere reorganization in some taxa are discussed.     

Telomeric repeat sequences

Chromosomes not only carry transcribed genes and their regulatory DNA sequences, but also contain regions that are required for the stability and maintenace of the chromosome as a unit. These include centromeres, telomeres and origins of replication. It is clear for replication origins and centromeres that the positions of these chromosomal organelles are determined by sites of the appropriate DNA sequences, but also that functional performance requires one or more contributing proteins. Telomeres are also structurally complex, with one or more DNA components, including simple telomeric repeats and more complex telomere-associated sequences, as well as one or more specific proteins that recognize these sequences. Accumulating evidence suggests that the simple telomeric repeats are required in most, but not all species, although they are not sufficient to determine the chromosomal position of a telomere.     

Constraints in chromosomal inversions in Escherichia coli are not explained by replication pausing at inverted terminator-like sequences

Regions close to the replication terminus of the Escherichia coli chromosome are strongly refractory to genomic inversions. Since these regions also harbour polar replication terminator-like sequences or pause sites, we have investigated the possibility that slowing of replication as a result of pausing at inverted pause sites is responsible for inability to isolate stable inversions affecting these regions. A mutation in the tus gene is known to abolish replication pausing at terminators. We show here that the distribution of invertible and noninvertible segments along the chromosome is not affected by tus mutations. This observation eliminates replication pausing as a cause for the reduced fitness of bacteria harbouring certain chromosomal inversions.     

Description of a chromosome replication unit in individual prematurely condensed human S-phase chromosomes

Mammalian chromosome replication was studied by the aid of premature chromosome condensation (PCC). After induction of PCC the sites of DNA replication appear as “gaps” between condensed chromosomal regions. These condensed particles are unineme before and bineme after DNA replication. The two phases are due mainly to the unineme or bineme nature of the particles. During early S-phase almost all particles are unineme, during late S-phase they are bineme and there is only one transitory stage between these two main stages. Premature chromosome condensation was studied in detail on a specific human chromosome 22 which is marked by its heterochromatin constitution. This led to easy identification of these elements in S-phase PCC (S-PCC) preparations. For each stage of the S-phase there was a reproducible pattern of condensed chromosomal particles making up the whole chromosome. The number of these particles was rather limited and a complementary pattern was found in early versus late S-phase. The pattern of early S-PCC corresponded to the banding pattern of G-banded prometaphase chromosomes; the pattern of late S-PCC, to R-banded prometaphase chromosomes. Thus, “gaps” and condensed particles as observed after PCC induction are obviously homologous to chromosome replication units. Replication of constitutive heterochromatin occurred during the very late S-phase. During this stage PCC induction led to condensation of the heterochromatin into several small, highly fluorescent particles.