RIF1 Links Replication Timing with Fork Reactivation and DNA Double-Strand Break Repair.

Replication timing (RT) is a cellular program to coordinate initiation of DNA replication in all origins within the genome. RIF1 (replication timing regulatory factor 1) is a master regulator of RT in human cells. This role of RIF1 is associated with binding G4-quadruplexes and changes in 3D chromatin that may suppress origin activation over a long distance. Many effects of RIF1 in fork reactivation and DNA double-strand (DSB) repair (DSBR) are underlined by its interaction with TP53BP1 (tumor protein p53 binding protein). In G1, RIF1 acts antagonistically to BRCA1 (BRCA1 DNA repair associated), suppressing end resection and homologous recombination repair (HRR) and promoting non-homologous end joining (NHEJ), contributing to DSBR pathway choice. RIF1 is an important element of intra-S-checkpoints to recover damaged replication fork with the involvement of HRR. High-resolution microscopic studies show that RIF1 cooperates with TP53BP1 to preserve 3D structure and epigenetic markers of genomic loci disrupted by DSBs. Apart from TP53BP1, RIF1 interact with many other proteins, including proteins involved in DNA damage response, cell cycle regulation, and chromatin remodeling. As impaired RT, DSBR and fork reactivation are associated with genomic instability, a hallmark of malignant transformation, RIF1 has a diagnostic, prognostic, and therapeutic potential in cancer. Further studies may reveal other aspects of common regulation of RT, DSBR, and fork reactivation by RIF1.

Low Replicative Stress Triggers Cell-Type Specific Inheritable Advanced Replication Timing.

DNA replication timing (RT), reflecting the temporal order of origin activation, is known as a robust and conserved cell-type specific process. Upon low replication stress, the slowing of replication forks induces well-documented RT delays associated to genetic instability, but it can also generate RT advances that are still uncharacterized. In order to characterize these advanced initiation events, we monitored the whole genome RT from six independent human cell lines treated with low doses of aphidicolin. We report that RT advances are cell-type-specific and involve large heterochromatin domains. Importantly, we found that some major late to early RT advances can be inherited by the unstressed next-cellular generation, which is a unique process that correlates with enhanced chromatin accessibility, as well as modified replication origin landscape and gene expression in daughter cells. Collectively, this work highlights how low replication stress may impact cellular identity by RT advances events at a subset of chromosomal domains.

Genome-wide mapping of human DNA replication by optical replication mapping supports a stochastic model of eukaryotic replication.

The heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation sites make mapping the location and timing of replication initiation in human cells difficult. To address this challenge, we have developed optical replication mapping (ORM), a high-throughput single-molecule approach, and used it to map early-initiation events in human cells. The single-molecule nature of our data and a total of >2,500-fold coverage of the human genome on 27 million fibers averaging ∼300 kb in length allow us to identify initiation sites and their firing probability with high confidence. We find that the distribution of human replication initiation is consistent with inefficient, stochastic activation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.

3D genome organization contributes to genome instability at fragile sites.

Common fragile sites (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features were suggested to underlie CFS instability, however, these features are prevalent across the genome. Therefore, the molecular mechanisms underlying CFS instability remain unclear. Here, we explore the transcriptional profile and DNA replication timing (RT) under mild replication stress in the context of the 3D genome organization. The results reveal a fragility signature, comprised of a TAD boundary overlapping a highly transcribed large gene with APH-induced RT-delay. This signature enables precise mapping of core fragility regions in known CFSs and identification of novel fragile sites. CFS stability may be compromised by incomplete DNA replication and repair in TAD boundaries core fragility regions leading to genomic instability. The identified fragility signature will allow for a more comprehensive mapping of CFSs and pave the way for investigating mechanisms promoting genomic instability in cancer.

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.

Genome-wide high-resolution mapping of mitotic DNA synthesis sites and common fragile sites by direct sequencing.

Common fragile sites (CFSs) are genomic loci prone to the formation of breaks or gaps on metaphase chromosomes. They are hotspots for chromosome rearrangements and structural variations, which have been extensively implicated in carcinogenesis, aging, and other pathological processes. Although many CFSs were identified decades ago, a consensus is still lacking for why they are particularly unstable and sensitive to replication perturbations. This is in part due to the lack of high-resolution mapping data for the vast majority of the CFSs, which has hindered mechanistic interrogations. Here, we seek to map human CFSs with high resolution on a genome-wide scale by sequencing the sites of mitotic DNA synthesis (MiDASeq) that are specific for CFSs. We generated a nucleotide-resolution atlas of MiDAS sites (MDSs) that covered most of the known CFSs, and comprehensively analyzed their sequence characteristics and genomic features. Our data on MDSs tallied well with long-standing hypotheses to explain CFS fragility while highlighting the contributions of late replication timing and large transcription units. Notably, the MDSs also encompassed most of the recurrent double-strand break clusters previously identified in mouse neural stem/progenitor cells, thus bridging evolutionarily conserved break points across species. Moreover, MiDAseq provides an important resource that can stimulate future research on CFSs to further unravel the mechanisms and biological relevance underlying these labile genomic regions.

Transcription-mediated organization of the replication initiation program across large genes sets common fragile sites genome-wide.

Common fragile sites (CFSs) are chromosome regions prone to breakage upon replication stress known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription could elicit their instability; however, the underlying mechanisms remain elusive. Genome-wide replication timing analyses here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, replicated by long-travelling forks. Forks that travel long in late S phase explains CFS replication features, whereas formation of sequence-dependent fork barriers or head-on transcription-replication conflicts do not. We further show that transcription inhibition during S phase, which suppresses transcription-replication encounters and prevents origin resetting, could not rescue CFS stability. Altogether, our results show that transcription-dependent suppression of initiation events delays replication of large gene bodies, committing them to instability.

Germline DNA replication timing shapes mammalian genome composition.

Mammalian DNA replication is a highly organized and regulated process. Large, Mb-sized regions are replicated at defined times along S-phase. Replication Timing (RT) is thought to play a role in shaping the mammalian genome by affecting mutation rates. Previous analyses relied on somatic RT profiles. However, only germline mutations are passed on to offspring and affect genomic composition. Therefore, germ cell RT information is necessary to evaluate the influences of RT on the mammalian genome. We adapted the RT mapping technique for limited amounts of cells, and measured RT from two stages in the mouse germline - primordial germ cells (PGCs) and spermatogonial stem cells (SSCs). RT in germline cells exhibited stronger correlations to both mutation rate and recombination hotspots density than those of RT in somatic tissues, emphasizing the importance of using correct tissues-of-origin for RT profiling. Germline RT maps exhibited stronger correlations to additional genetic features including GC-content, transposable elements (SINEs and LINEs), and gene density. GC content stratification and multiple regression analysis revealed independent contributions of RT to SINE, gene, mutation, and recombination hotspot densities. Together, our results establish a central role for RT in shaping multiple levels of mammalian genome composition.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

PREP1 tumor suppressor protects the late-replicating DNA by controlling its replication timing and symmetry.

The synthesis of middle-to-late-replicating DNA can be affected independently of the rest of the genome by down-regulating the tumor suppressor PREP1 (PKNOX1). Indeed, DNA combing shows that PREP1 down-regulation affects DNA replication rate, increases the number of simultaneously firing origins and the asymmetry of DNA replication, leading to DNA damage. Genome-wide analysis of replication timing by Repli-seq shows that, upon PREP1 down-regulation, 25% of the genome is replicated earlier in the S-phase. The targeted DNA sequences correspond to Lamin-Associated Domains (LADs), and include late-replicating (LRRs) and temporal transition regions (TTRs). Notably, the distribution of PREP1 DNA binding sites and of its target genes indicates that DNA replication defects are independent of the overall PREP1 transcriptional activity. Finally, PREP1 down-regulation causes a substantial decrease in Lamin B1 levels. This suggests that DNA is released from the nuclear lamina earlier than in the control cells and is available for replication, thus explaining timing defects and DNA damage.This is the first evidence that the replication timing of a specific fraction of the human genome is affected by PREP1 tumor suppressor. This previously unknown function might significantly contribute to the genomic instability observed in human tumors.

Differential DNA mismatch repair underlies mutation rate variation across the human genome.

Cancer genome sequencing has revealed considerable variation in somatic mutation rates across the human genome, with mutation rates elevated in heterochromatic late replicating regions and reduced in early replicating euchromatin. Multiple mechanisms have been suggested to underlie this, but the actual cause is unknown. Here we identify variable DNA mismatch repair (MMR) as the basis of this variation. Analysing ∼17 million single-nucleotide variants from the genomes of 652 tumours, we show that regional autosomal mutation rates at megabase resolution are largely stable across cancer types, with differences related to changes in replication timing and gene expression. However, mutations arising after the inactivation of MMR are no longer enriched in late replicating heterochromatin relative to early replicating euchromatin. Thus, differential DNA repair and not differential mutation supply is the primary cause of the large-scale regional mutation rate variation across the human genome.

G4 motifs affect origin positioning and efficiency in two vertebrate replicators.

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome-wide studies in vertebrates have recently identified a consensus G-rich motif potentially able to form G-quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.

DNA replication timing and higher-order nuclear organization determine single-nucleotide substitution patterns in cancer genomes.

Single-nucleotide substitutions are a defining characteristic of cancer genomes. Many single-nucleotide substitutions in cancer genomes arise because of errors in DNA replication, which is spatio-temporally stratified. Here we propose that DNA replication patterns help shape the mutational landscapes of normal and cancer genomes. Using data on five fully sequenced cancer types and two personal genomes, we determined that the frequency of intergenic single-nucleotide substitution is significantly higher in late DNA replication timing regions, even after controlling for a number of genomic features. Furthermore, some substitution signatures are more frequent in certain DNA replication timing zones. Finally, integrating data on higher-order nuclear organization, we found that genomic regions in close spatial proximity to late-replicating domains display similar mutation spectra as the late-replicating regions themselves. These data suggest that DNA replication timing together with higher-order genomic organization contribute to the patterns of single-nucleotide substitution in normal and cancer genomes.

DNA replication timing, genome stability and cancer: late and/or delayed DNA replication timing is associated with increased genomic instability.

Normal cellular division requires that the genome be faithfully replicated to ensure that unaltered genomic information is passed from one generation to the next. DNA replication initiates from thousands of origins scattered throughout the genome every cell cycle; however, not all origins initiate replication at the same time. A vast amount of work over the years indicates that different origins along each eukaryotic chromosome are activated in early, middle or late S phase. This temporal control of DNA replication is referred to as the replication-timing program. The replication-timing program represents a very stable epigenetic feature of chromosomes. Recent evidence has indicated that the replication-timing program can influence the spatial distribution of mutagenic events such that certain regions of the genome experience increased spontaneous mutagenesis compared to surrounding regions. This influence has helped shape the genomes of humans and other multicellular organisms and can affect the distribution of mutations in somatic cells. It is also becoming clear that the replication-timing program is deregulated in many disease states, including cancer. Aberrant DNA replication timing is associated with changes in gene expression, changes in epigenetic modifications and an increased frequency of structural rearrangements. Furthermore, certain replication timing changes can directly lead to overt genomic instability and may explain unique mutational signatures that are present in cells that have undergone the recently described processes of "chromothripsis" and "kataegis". In this review, we will discuss how the normal replication timing program, as well as how alterations to this program, can contribute to the evolution of the genomic landscape in normal and cancerous cells.

Alterations in replication timing of cancer-related genes in malignant human breast cancer cells.

The replication timing of nine genes commonly involved in cancer was investigated in the MCF10 cell lines for human breast cancer progression. Six of these nine genes are part of a constellation of tumor suppressor genes that play a major role in familial human breast cancer (TP53, ATM, PTEN, CHK2, BRCA1, and BRCA2). Three other genes are involved in a large number of human cancers including breast as either tumor suppressors (RB1 and RAD51) or as an oncogene (cMYC). Five of these nine genes (TP53, RAD51, ATM, PTEN, and cMYC) show significant differences (P < 0.05) in replication timing between MCF10A normal human breast cells and the corresponding malignant MCF10CA1a cells. These differences are specific to the malignant state of the MCF10CA1a cells since there were no significant differences in the replication timing of these genes between normal MCF10A cells and the non-malignant cancer MCF10AT1 cells. Microarray analysis further demonstrated that three of these five genes (TP53, RAD51, and cMYC) showed significant changes in gene expression (≥2-fold) between normal and malignant cells. Our findings demonstrate an alteration in the replication timing of a small subset of cancer-related genes in malignant breast cancer cells. These alterations partially correlate with the major transcriptional changes characteristic of the malignant state in these cells.

DNA replication timing and long-range DNA interactions predict mutational landscapes of cancer genomes.

Somatic copy-number alterations (SCNA) are a hallmark of many cancer types, but the mechanistic basis underlying their genome-wide patterns remains incompletely understood. Here we integrate data on DNA replication timing, long-range interactions between genomic material, and 331,724 SCNAs from 2,792 cancer samples classified into 26 cancer types. We report that genomic regions of similar replication timing are clustered spatially in the nucleus, that the two boundaries of SCNAs tend to be found in such regions, and that regions replicated early and late display distinct patterns of frequencies of SCNA boundaries, SCNA size and a preference for deletions over insertions. We show that long-range interaction and replication timing data alone can identify a significant proportion of SCNAs in an independent test data set. We propose a model for the generation of SCNAs in cancer, suggesting that data on spatial proximity of regions replicating at the same time can be used to predict the mutational landscapes of cancer genomes.

Replication timing aberrations and aneuploidy in peripheral blood lymphocytes of breast cancer patients.

BACKGROUND: Peripheral blood lymphocytes of patients with hematological malignancies or solid tumors, such as renal cell carcinoma or prostate cancer, display epigenetic aberrations (loss of synchronous replication of allelic counterparts) and genetic changes (aneuploidy) characteristic of the cancerous phenotype. This study sought to determine whether such alterations could differentiate breast cancer patients from cancer-free subjects. METHODS: The HER2 locus-an oncogene assigned to chromosome 17 whose amplification is associated with breast cancer (BCA)-and the pericentromeric satellite sequence of chromosome 17 (CEN17) were used for replication timing assessments. Aneuploidy was monitored by enumerating the copy numbers of chromosome 17. Replication timing and aneuploidy were detected cytogenetically using fluorescence in situ hybridization technology applied to phytohemagglutinin-stimulated lymphocytes of 20 women with BCA and 10 control subjects. RESULTS: We showed that both the HER2 and CEN17 loci in the stimulated BCA lymphocytes altered their characteristic pattern of synchronous replication and exhibited asynchronicity. In addition, there was an increase in chromosome 17 aneuploidy. The frequency of cells displaying asynchronous replication in the patients' samples was significantly higher (P < 10(-12) for HER2 and P < 10(-6) for CEN17) than the corresponding values in the control samples. Similarly, aneuploidy in patients' cells was significantly higher (P < 10(-9)) than that in the controls. CONCLUSIONS: The HER2 and CEN17 aberrant replication differentiated clearly between BCA patients and control subjects. Thus, monitoring the replication of these genes offers potential blood markers for the detection and monitoring of breast cancer.