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"ORIGINS"
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The Neolithic revolution
This book examines the factors that could have led to this revolution and the archaeological evidence of which changes happened where and when.
Book
Establishment and function of chromatin organization at replication origins
The origin recognition complex (ORC) is essential for initiation of eukaryotic chromosome replication as it loads the replicative helicase—the minichromosome maintenance (MCM) complex—at replication origins
1
. Replication origins display a stereotypic nucleosome organization with nucleosome depletion at ORC-binding sites and flanking arrays of regularly spaced nucleosomes
2
–
4
. However, how this nucleosome organization is established and whether this organization is required for replication remain unknown. Here, using genome-scale biochemical reconstitution with approximately 300 replication origins, we screened 17 purified chromatin factors from budding yeast and found that the ORC established nucleosome depletion over replication origins and flanking nucleosome arrays by orchestrating the chromatin remodellers INO80, ISW1a, ISW2 and Chd1. The functional importance of the nucleosome-organizing activity of the ORC was demonstrated by
orc1
mutations that maintained classical MCM-loader activity but abrogated the array-generation activity of ORC. These mutations impaired replication through chromatin in vitro and were lethal in vivo. Our results establish that ORC, in addition to its canonical role as the MCM loader, has a second crucial function as a master regulator of nucleosome organization at the replication origin, a crucial prerequisite for efficient chromosome replication.
Genome-scale in vitro reconstitution of DNA replication through chromatin establishes a crucial role for the origin recognition complex in organizing nucleosome arrays that are crucial for the initiation of replication.
Journal Article
The Invention of Tradition
Many of the traditions which we think of as very ancient in their origins were not in fact sanctioned by long usage over the centuries, but were invented comparatively recently. This book explores examples of this process of invention – the creation of Welsh and Scottish 'national culture'; the elaboration of British royal rituals in the nineteenth and twentieth centuries; the origins of imperial rituals in British India and Africa; and the attempts by radical movements to develop counter-traditions of their own. It addresses the complex interaction of past and present, bringing together historians and anthropologists in a fascinating study of ritual and symbolism which poses new questions for the understanding of our history.
eBook
Replication landscape of the human genome
Despite intense investigation, human replication origins and termini remain elusive. Existing data have shown strong discrepancies. Here we sequenced highly purified Okazaki fragments from two cell types and, for the first time, quantitated replication fork directionality and delineated initiation and termination zones genome-wide. Replication initiates stochastically, primarily within non-transcribed, broad (up to 150 kb) zones that often abut transcribed genes, and terminates dispersively between them. Replication fork progression is significantly co-oriented with the transcription. Initiation and termination zones are frequently contiguous, sometimes separated by regions of unidirectional replication. Initiation zones are enriched in open chromatin and enhancer marks, even when not flanked by genes, and often border ‘topologically associating domains’ (TADs). Initiation zones are enriched in origin recognition complex (ORC)-binding sites and better align to origins previously mapped using bubble-trap than λ-exonuclease. This novel panorama of replication reveals how chromatin and transcription modulate the initiation process to create cell-type-specific replication programs.
The physical origin and termination sites of DNA replication in human cells have remained elusive. Here the authors use Okazaki fragment sequencing to reveal global replication patterns and show how chromatin and transcription modulate the process.
Journal Article
Cohesin-mediated loop anchors confine the locations of human replication origins
DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability
1
,
2
. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)
3
–
6
, subTADs
7
and loops
8
in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.
A study shows that the three-dimensional conformation of the human genome influences the positioning of DNA replication initiation zones, highlighting cohesin-mediated loop anchors as essential determinants of their precise location.
Journal Article
Structure of the origin recognition complex bound to DNA replication origin
The six-subunit origin recognition complex (ORC) binds to DNA to mark the site for the initiation of replication in eukaryotes. Here we report a 3 Å cryo-electron microscopy structure of the
Saccharomyces cerevisiae
ORC bound to a 72-base-pair origin DNA sequence that contains the ARS consensus sequence (ACS) and the B1 element. The ORC encircles DNA through extensive interactions with both phosphate backbone and bases, and bends DNA at the ACS and B1 sites. Specific recognition of thymine residues in the ACS is carried out by a conserved basic amino acid motif of Orc1 in the minor groove, and by a species-specific helical insertion motif of Orc4 in the major groove. Moreover, similar insertions into major and minor grooves are also embedded in the B1 site by basic patch motifs from Orc2 and Orc5, respectively, to contact bases and to bend DNA. This work pinpoints a conserved role of ORC in modulating DNA structure to facilitate origin selection and helicase loading in eukaryotes.
The cryo-EM structure of the yeast origin recognition complex (ORC) bound to a 72-base-pair origin DNA sequence provides insights into the basis of the origin selection mechanism.
Journal Article
Bidirectional eukaryotic DNA replication is established by quasi-symmetrical helicase loading
Chromosomal DNA replication initiates bidirectionally by loading two ring-shaped helicases onto DNA in opposite orientations. How this symmetry is achieved has been puzzling because replication initiation sites contain only one essential binding site for the initiator, the origin recognition complex (ORC). Coster and Diffley now show that both helicases are loaded by a similar mechanism. Efficient loading requires binding of two ORC complexes to two ORC binding sites in opposite orientations. Natural origins were found to be partially symmetrical, containing functionally relevant secondary ORC sites. Sites can be flexibly spaced, but introducing an intervening “roadblock” prevented loading, suggesting that individual helicases translocate toward each other on DNA to form a stable double ring. Science , this issue p. 314 The minichromosome maintenance double hexamer, a precursor of the replicative DNA helicase, results from a symmetrical loading reaction. Bidirectional replication from eukaryotic DNA replication origins requires the loading of two ring-shaped minichromosome maintenance (MCM) helicases around DNA in opposite orientations. MCM loading is orchestrated by binding of the origin recognition complex (ORC) to DNA, but how ORC coordinates symmetrical MCM loading is unclear. We used natural budding yeast DNA replication origins and synthetic DNA sequences to show that efficient MCM loading requires binding of two ORC molecules to two ORC binding sites. The relative orientation of these sites, but not the distance between them, was found to be critical for MCM loading in vitro and origin function in vivo. We propose that quasi-symmetrical loading of individual MCM hexamers by ORC and directed MCM translocation into double hexamers acts as a unifying mechanism for the establishment of bidirectional replication in archaea and eukaryotes.
Journal Article
MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing
Origin recognition complex (ORC)-dependent loading of the replicative helicase MCM2-7 onto replication origins in G1-phase forms the basis of replication fork establishment in S-phase. However, how ORC and MCM2-7 facilitate genome-wide DNA licensing is not fully understood. Mapping the molecular footprints of budding yeast ORC and MCM2-7 genome-wide, we discovered that MCM2-7 loading is associated with ORC release from origins and redistribution to non-origin sites. Our bioinformatic analysis revealed that origins are compact units, where a single MCM2-7 double hexamer blocks repetitive loading through steric ORC binding site occlusion. Analyses of A-elements and an improved B2-element consensus motif uncovered that DNA shape, DNA flexibility, and the correct, face-to-face spacing of the two DNA elements are hallmarks of ORC-binding and efficient helicase loading sites. Thus, our work identified fundamental principles for MCM2-7 helicase loading that explain how origin licensing is realised across the genome.
Correct loading of the MCM2-7 helicase is crucial for DNA replication and cell cycle progression. Here, the authors used high-resolution genomics to demonstrate how ORC is displaced from origins, which serves as a mechanism for distributive MCM loading onto DNA.
Journal Article
Nucleosome-directed replication origin licensing independent of a consensus DNA sequence
The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast
Saccharomyces cerevisiae
(S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that
Xenopus laevis
nucleosomes can substitute for yeast ones in engaging with ORC. Combined with re-analyses of genome-wide ORC binding data, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.
Most eukaryotes do not use a consensus DNA sequence as binding sites for the origin recognition complex (ORC) to initiate DNA replication, however budding yeast do. Here the authors show S. cerevisiae ORC can bind nucleosomes near nucleosome-free regions and recruit replicative helicases to form a pre-replication complex independent of the DNA sequence.
Journal Article
Integrative analysis of DNA replication origins and ORC-/MCM-binding sites in human cells reveals a lack of overlap
Based on experimentally determined average inter-origin distances of ~100 kb, DNA replication initiates from ~50,000 origins on human chromosomes in each cell cycle. The origins are believed to be specified by binding of factors like the origin recognition complex (ORC) or CTCF or other features like G-quadruplexes. We have performed an integrative analysis of 113 genome-wide human origin profiles (from five different techniques) and five ORC-binding profiles to critically evaluate whether the most reproducible origins are specified by these features. Out of ~7.5 million union origins identified by all datasets, only 0.27% (20,250 shared origins) were reproducibly obtained in at least 20 independent SNS-seq datasets and contained in initiation zones identified by each of three other techniques, suggesting extensive variability in origin usage and identification. Also, 21% of the shared origins overlap with transcriptional promoters, posing a conundrum. Although the shared origins overlap more than union origins with constitutive CTCF-binding sites, G-quadruplex sites, and activating histone marks, these overlaps are comparable or less than that of known transcription start sites, so that these features could be enriched in origins because of the overlap of origins with epigenetically open, promoter-like sequences. Only 6.4% of the 20,250 shared origins were within 1 kb from any of the ~13,000 reproducible ORC-binding sites in human cancer cells, and only 4.5% were within 1 kb of the ~11,000 union MCM2-7-binding sites in contrast to the nearly 100% overlap in the two comparisons in the yeast, Saccharomyces cerevisiae . Thus, in human cancer cell lines, replication origins appear to be specified by highly variable stochastic events dependent on the high epigenetic accessibility around promoters, without extensive overlap between the most reproducible origins and currently known ORC- or MCM-binding sites.
Journal Article