Genome-wide mapping of human DNA replication by optical replication mapping supports a stochastic model of eukaryotic replication.
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ABSTRACT:
SUBMITTER: Wang W
PROVIDER: S-EPMC8286344 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:To investigate DNA replication enzymology across the nuclear genome of budding yeast, deep sequencing was used to establish the pattern of uncorrected replication errors generated by an asymmetric mutator variant of DNA polymerase ? (Pol ?). Sequencing of 16 genomes identified 1,206-bp substitutions generated over 33 generations by L612M Pol ? in a mismatch repair defective strain. Alignment of sequences flanking these substitutions identified "hotspot" motifs for Pol ? replication errors. The substitutions were distributed evenly across all 16 chromosomes. The vast majority were transitions that occurred with a strand bias that varied in a predictable manner relative to known functional origins of replication. This strand bias strongly supports the idea that Pol ? is primarily a lagging strand polymerase during replication across the entire nuclear genome.
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Project description:Methylation of CpG dinucleotides is a fundamental mechanism of epigenetic regulation in eukaryotic genomes. Development of methods for rapid genome wide methylation profiling will greatly facilitate both hypothesis and discovery driven research in the field of epigenetics. In this regard, a single molecule approach to methylation profiling offers several unique advantages that include elimination of chemical DNA modification steps and PCR amplification.A single molecule approach is presented for the discernment of methylation profiles, based on optical mapping. We report results from a series of pilot studies demonstrating the capabilities of optical mapping as a platform for methylation profiling of whole genomes. Optical mapping was used to discern the methylation profile from both an engineered and wild type Escherichia coli. Furthermore, the methylation status of selected loci within the genome of human embryonic stem cells was profiled using optical mapping.The optical mapping platform effectively detects DNA methylation patterns. Due to single molecule detection, optical mapping offers significant advantages over other technologies. This advantage stems from obviation of DNA modification steps, such as bisulfite treatment, and the ability of the platform to assay repeat dense regions within mammalian genomes inaccessible to techniques using array-hybridization technologies.
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Project description:BACKGROUND:DNA replication initiates on defined genome sites, termed origins. Origin usage appears to follow common rules in the eukaryotic organisms examined to date: all chromosomes are replicated from multiple origins, which display variations in firing efficiency and are selected from a larger pool of potential origins. To ask if these features of DNA replication are true of all eukaryotes, we describe genome-wide origin mapping in the parasite Leishmania. RESULTS:Origin mapping in Leishmania suggests a striking divergence in origin usage relative to characterized eukaryotes, since each chromosome appears to be replicated from a single origin. By comparing two species of Leishmania, we find evidence that such origin singularity is maintained in the face of chromosome fusion or fission events during evolution. Mapping Leishmania origins suggests that all origins fire with equal efficiency, and that the genomic sites occupied by origins differ from related non-origins sites. Finally, we provide evidence that origin location in Leishmania displays striking conservation with Trypanosoma brucei, despite the latter parasite replicating its chromosomes from multiple, variable strength origins. CONCLUSIONS:The demonstration of chromosome replication for a single origin in Leishmania, a microbial eukaryote, has implications for the evolution of origin multiplicity and associated controls, and may explain the pervasive aneuploidy that characterizes Leishmania chromosome architecture.
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Project description:Eukaryotic DNA replication origins differ both in their efficiency and in the characteristic time during S phase when they become active. The biological basis for these differences remains unknown, but they could be a consequence of chromatin structure. The availability of genome-wide maps of nucleosome positions has led to an explosion of information about how nucleosomes are assembled at transcription start sites, but no similar maps exist for DNA replication origins. Here we combine high-resolution genome-wide nucleosome maps with comprehensive annotations of DNA replication origins to identify patterns of nucleosome occupancy at eukaryotic replication origins. On average, replication origins contain a nucleosome depleted region centered next to the ACS element, flanked on both sides by arrays of well-positioned nucleosomes. Our analysis identified DNA sequence properties that correlate with nucleosome occupancy at replication origins genome-wide and that are correlated with the nucleosome-depleted region. Clustering analysis of all annotated replication origins revealed a surprising diversity of nucleosome occupancy patterns. We provide evidence that the origin recognition complex, which binds to the origin, acts as a barrier element to position and phase nucleosomes on both sides of the origin. Finally, analysis of chromatin reconstituted in vitro reveals that origins are inherently nucleosome depleted. Together our data provide a comprehensive, genome-wide view of chromatin structure at replication origins and suggest a model of nucleosome positioning at replication origins in which the underlying sequence occludes nucleosomes to permit binding of the origin recognition complex, which then (likely in concert with nucleosome modifiers and remodelers) positions nucleosomes adjacent to the origin to promote replication origin function.
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Project description:Mechanisms regulating where and when eukaryotic DNA replication initiates remain a mystery. Recently, genome-scale methods have been brought to bear on this problem. The identification of replication origins and their associated proteins in yeasts is a well-integrated investigative tool, but corresponding data sets from multicellular organisms are scarce. By contrast, standardized protocols for evaluating replication timing have generated informative data sets for most eukaryotic systems. Here, I summarize the genome-scale methods that are most frequently used to analyse replication in eukaryotes, the kinds of questions each method can address and the technical hurdles that must be overcome to gain a complete understanding of the nature of eukaryotic replication origins.
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Project description:We describe a genome-wide DNA double-strand break (DSB) mapping technique, Break-seq. In this protocol, we provide step-by-step instructions for cell embedment in agarose, in-gel DSB labeling and subsequent capture, followed by standard Illumina library construction and sequencing. We also provide the framework for sequence data processing and DSB peak identification. Finally, we present a custom-designed 3D-printed device for processing agarose-embedded DNA samples. The protocol is applicable to Saccharomyces cerevisiae, as well as mammalian suspension, adherent, and 3D organoid cell cultures. For complete details on the use and execution of this protocol, please refer to Hoffman et al. (2015) and Chakraborty et al. (2020).
| S-EPMC8220103 | biostudies-literature
Project description:DNA replication in eukaryotic cells initiates from hundreds of origins along their genomes, leading to complete duplication of genetic information before cell division. The large number of potential origins, coupled with system uncertainty, dictates the need for new analytical tools to capture spatial and temporal patterns of DNA replication genome-wide. We have developed a stochastic hybrid model that reproduces DNA replication throughout a complete genome. The model can capture different modes of DNA replication and is applicable to various organisms. Using genome-wide data on the location and firing efficiencies of origins in the fission yeast, we show how the DNA replication process evolves during S-phase in the presence of stochastic origin firing. Simulations reveal small regions of the genome that extend S-phase to three times its reported duration. The low levels of late replication predicted by the model are below the detection limit of techniques used to measure S-phase length. Parameter sensitivity analysis shows that increased replication fork speeds genome-wide, or additional origins are not sufficient to reduce S-phase to its reported length. We model the redistribution of a limiting initiation factor during S-phase and show that it could shorten S-phase to the reported duration. Alternatively, S-phase may be extended, and what has traditionally been defined as G2 may be occupied by low levels of DNA synthesis with the onset of mitosis delayed by activation of the G2/M checkpoint.
| S-EPMC2527905 | biostudies-other
Project description:Many fundamental aspects of DNA replication, such as the exact locations where DNA synthesis is initiated and terminated, how frequently origins are used, and how fork progression is influenced by transcription, are poorly understood. Via the deep sequencing of Okazaki fragments, we comprehensively document replication fork directionality throughout the S. cerevisiae genome, which permits the systematic analysis of initiation, origin efficiency, fork progression, and termination. We show that leading-strand initiation preferentially occurs within a nucleosome-free region at replication origins. Using a strain in which late origins can be induced to fire early, we show that replication termination is a largely passive phenomenon that does not rely on cis-acting sequences or replication fork pausing. The replication profile is predominantly determined by the kinetics of origin firing, allowing us to reconstruct chromosome-wide timing profiles from an asynchronous culture.
| S-EPMC3628276 | biostudies-literature
Project description:The heterozygous diploid genome of Candida albicans displays frequent genomic rearrangements, in particular loss-of-heterozygosity (LOH) events, which can be seen on all eight chromosomes and affect both laboratory and clinical strains. LOHs, which are often the consequence of DNA damage repair, can be observed upon stresses reminiscent of the host environment, and result in homozygous regions of various sizes depending on the molecular mechanisms at their origins. Recent studies have shed light on the biological importance of these frequent and ubiquitous LOH events in C. albicans. In diploid Saccharomyces cerevisiae, LOH facilitates the passage of recessive beneficial mutations through Haldane's sieve, allowing rapid evolutionary adaptation. This also appears to be true in C. albicans, where the full potential of an adaptive mutation is often only observed upon LOH, as illustrated in the case of antifungal resistance and niche adaptation. To understand the genome-wide dynamics of LOH events in C. albicans, we constructed a collection of 15 strains, each one carrying a LOH reporter system on a different chromosome arm. This system involves the insertion of two fluorescent marker genes in a neutral genomic region on both homologs, allowing spontaneous LOH events to be detected by monitoring the loss of one of the fluorescent markers using flow cytometry. Using this collection, we observed significant LOH frequency differences between genomic loci in standard laboratory growth conditions; however, we further demonstrated that comparable heterogeneity was also observed for a given genomic locus between independent strains. Additionally, upon exposure to stress, three outcomes could be observed in C. albicans, where individual strains displayed increases, decreases, or no effect of stress in terms of LOH frequency. Our results argue against a general stress response triggering overall genome instability. Indeed, we showed that the heterogeneity of LOH frequency in C. albicans is present at various levels, inter-strain, intra-strain, and inter-chromosomes, suggesting that LOH events may occur stochastically within a cell, though the genetic background potentially impacts genome stability in terms of LOH throughout the genome in both basal and stress conditions. This heterogeneity in terms of genome stability may serve as an important adaptive strategy for the predominantly clonal human opportunistic pathogen C. albicans, by quickly generating a wide spectrum of genetic variation combinations potentially permitting subsistence in a rapidly evolving environment.
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Project description:To maintain genomic stability, reinitiation of eukaryotic DNA replication within a single cell cycle is blocked by multiple mechanisms that inactivate or remove replication proteins after G1 phase. Consistent with the prevailing notion that these mechanisms are redundant, we previously showed that simultaneous deregulation of three replication proteins, ORC, Cdc6, and Mcm2-7, was necessary to cause detectable bulk re-replication in G2/M phase in Saccharomyces cerevisiae. In this study, we used microarray comparative genomic hybridization (CGH) to provide a more comprehensive and detailed analysis of re-replication. This genome-wide analysis suggests that reinitiation in G2/M phase primarily occurs at a subset of both active and latent origins, but is independent of chromosomal determinants that specify the use and timing of these origins in S phase. We demonstrate that re-replication can be induced within S phase, but differs in amount and location from re-replication in G2/M phase, illustrating the dynamic nature of DNA replication controls. Finally, we show that very limited re-replication can be detected by microarray CGH when only two replication proteins are deregulated, suggesting that the mechanisms blocking re-replication are not redundant. Therefore we propose that eukaryotic re-replication at levels below current detection limits may be more prevalent and a greater source of genomic instability than previously appreciated.
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