scholarly journals Germline DNA replication shapes the recombination landscape in mammals

2020 ◽  
Author(s):  
Florencia Pratto ◽  
Kevin Brick ◽  
Gang Cheng ◽  
Gabriel Lam ◽  
Jeffrey M. Cloutier ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Genetic Recombination ◽  
Chromosome Size ◽  
Replication Pattern ◽  
Replication Origins ◽  
Origin Firing ◽  
Recombination Hotspots ◽  
Novel Approach ◽  
Germline Variation

Summary:Genetic recombination generates novel trait combinations and understanding how recombination is distributed across the genome is key to modern genetics. The PRDM9 protein defines recombination hotspots, however megabase-scale recombination patterning is independent of PRDM9. The single round of DNA replication, which precedes recombination in meiosis, may establish these patterns, therefore we devised a novel approach to study meiotic replication that includes robust and sensitive mapping of replication origins. We find that meiotic DNA replication is distinct; reduced origin firing slows replication in meiosis and a distinctive replication pattern in human males underlies the sub-telomeric increase in recombination. We detected a robust correlation between replication and both contemporary and ancestral recombination and found that replication origin density coupled with chromosome size determines the recombination potential of individual chromosomes. Our findings and methods have far-reaching implications for understanding the mechanisms underlying DNA replication, genetic recombination, and the landscape of mammalian germline variation.

scholarly journals Determination of human DNA replication origin position and efficiency reveals principles of initiation zone organisation

2021 ◽  
Author(s):  
Guillaume Guilbaud ◽  
Pierre Murat ◽  
Helen S Wilkes ◽  
Leticia Koch Lerner ◽  
Julian Sale ◽  
...  
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Gradient Centrifugation ◽  
Initiation Site ◽  
Efficiency Score ◽  
Replication Initiation ◽  
Equilibrium Density ◽  
Replication Origins ◽  
Origin Firing ◽  
Dna Replication Origin

Replication of the human genome initiates within broad zones of ~ 150 kb. The extent to which firing of individual DNA replication origins within initiation zones is spatially stochastic or localised at defined sites remains a matter of debate. A thorough characterisation of the dynamic activation of origins within initiation zones is hampered by the lack of a high-resolution map of both their position and efficiency. To address this shortcoming, we describe a modification of initiation site sequencing (ini-seq) based on density substitution. Newly-replicated DNA is rendered heavy-light (HL) by incorporation of BrdUTP, unreplicated DNA remaining light-light (LL). Replicated HL-DNA is separated from unreplicated LL-DNA by equilibrium density gradient centrifugation, then both fractions are subjected to massive parallel sequencing. This allows precise mapping of 23,905 replication origins simultaneously with an assignment of a replication initiation efficiency score to each. We show that origin firing within initiation zones is not randomly distributed. Rather, origins are arranged hierarchically with a set of very highly efficient origins marking zone boundaries. We propose that these origins explain much of the early firing activity arising within initiation zones, helping to unify the concept of replication initiation zones with the identification of discrete replication origin sites.

scholarly journals Transcription-coupled structural dynamics of topologically associating domains regulate replication origin efficiency

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yongzheng Li ◽  
Boxin Xue ◽  
Mengling Zhang ◽  
Liwei Zhang ◽  
Yingping Hou ◽  
...  
Keyword(s):  
Dna Replication ◽  
Structural Dynamics ◽  
Replication Origin ◽  
High Efficiency ◽  
S Phase ◽  
Chromatin Domain ◽  
Replication Origins ◽  
Replication Efficiency ◽  
Topologically Associating Domains ◽  
Epigenetic Signatures

Abstract Background Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Although various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined. Results We apply dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We find that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase. Intriguingly, while both high-efficiency and low-efficiency origins are distributed homogeneously in the TAD during the G1 phase, high-efficiency origins relocate to the TAD periphery before the S phase. Origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observe that the replication machinery protein PCNA forms immobile clusters around TADs at the G1/S transition, explaining why origins at the TAD periphery are preferentially fired. Conclusion Our work reveals a new origin selection mechanism that the replication efficiency of origins is determined by their physical distribution in the chromatin domain, which undergoes a transcription-dependent structural re-organization process. Our model explains the complex links between replication origin efficiency and many genetic and epigenetic signatures that mark active transcription. The coordination between DNA replication, transcription, and chromatin organization inside individual TADs also provides new insights into the biological functions of sub-domain chromatin structural dynamics.

scholarly journals Evolution of DNA replication origin specification and gene silencing mechanisms

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Y. Hu ◽  
A. Tareen ◽  
Y-J. Sheu ◽  
W. T. Ireland ◽  
C. Speck ◽  
...  
Keyword(s):  
Dna Replication ◽  
Gene Silencing ◽  
Dna Sequence ◽  
Origin Recognition Complex ◽  
Separate Analysis ◽  
Sequence Specificity ◽  
Replication Origins ◽  
Origin Firing ◽  
Dna Sequence Specificity ◽  
Α Helix

Abstract DNA replication in eukaryotic cells initiates from replication origins that bind the Origin Recognition Complex (ORC). Origin establishment requires well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove. Using a massively parallel origin selection assay coupled with a custom mutual-information-based modeling approach, and a separate analysis of whole-genome replication profiling, here we show that the Orc4 α-helix contributes to the DNA sequence-specificity of origins in S. cerevisiae and Orc4 α-helix mutations change genome-wide origin firing patterns. The DNA sequence specificity of replication origins, mediated by the Orc4 α-helix, has co-evolved with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.

scholarly journals DNA Replication Origins Fire Stochastically in Fission Yeast

2006 ◽  
Vol 17 (1) ◽  
pp. 308-316 ◽  
Author(s):  
Prasanta K. Patel ◽  
Benoit Arcangioli ◽  
Stephen P. Baker ◽  
Aaron Bensimon ◽  
Nicholas Rhind
Keyword(s):  
Dna Replication ◽  
Fission Yeast ◽  
Replication Initiation ◽  
Epigenetic Mechanism ◽  
Replication Origins ◽  
Origin Firing ◽  
Cell Cycles ◽  
Initiation Of Replication ◽  
Dna Combing ◽  
Dna Replication Origins

DNA replication initiates at discrete origins along eukaryotic chromosomes. However, in most organisms, origin firing is not efficient; a specific origin will fire in some but not all cell cycles. This observation raises the question of how individual origins are selected to fire and whether origin firing is globally coordinated to ensure an even distribution of replication initiation across the genome. We have addressed these questions by determining the location of firing origins on individual fission yeast DNA molecules using DNA combing. We show that the firing of replication origins is stochastic, leading to a random distribution of replication initiation. Furthermore, origin firing is independent between cell cycles; there is no epigenetic mechanism causing an origin that fires in one cell cycle to preferentially fire in the next. Thus, the fission yeast strategy for the initiation of replication is different from models of eukaryotic replication that propose coordinated origin firing.

scholarly journals Investigating the role of Rts1 in DNA replication initiation

2018 ◽  
Vol 3 ◽  
pp. 23 ◽  
Author(s):  
Ana B.A. Wallis ◽  
Conrad A. Nieduszynski
Keyword(s):  
Dna Replication ◽  
Temperature Sensitivity ◽  
Protein Phosphatase ◽  
Dna Helicase ◽  
Replication Initiation ◽  
Replication Origins ◽  
Origin Firing ◽  
Replication Factor ◽  
Dna Replication Initiation ◽  
Genomic Screen

Background: Understanding DNA replication initiation is essential to understand the mis-regulation of replication seen in cancer and other human disorders. DNA replication initiates from DNA replication origins. In eukaryotes, replication is dependent on cell cycle kinases which function during S phase. Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) act to phosphorylate the DNA helicase (composed of mini chromosome maintenance proteins: Mcm2-7) and firing factors to activate replication origins. It has recently been found that Rif1 can oppose DDK phosphorylation. Rif1 can recruit protein phosphatase 1 (PP1) to dephosphorylate MCM and restricts origin firing. In this study, we investigate a potential role for another phosphatase, protein phosphatase 2A (PP2A), in regulating DNA replication initiation. The PP2A regulatory subunit Rts1 was previously identified in a large-scale genomic screen to have a genetic interaction with ORC2 (a DNA replication licensing factor). Deletion of RTS1 synthetically rescued the temperature-sensitive (ts-) phenotype of ORC2 mutants. Methods: We deleted RTS1 in multiple ts-replication factor Saccharomyces cerevisiae strains, including ORC2.  Dilution series assays were carried out to compare qualitatively the growth of double mutant ∆rts1 ts-replication factor strains relative to the respective single mutant strains.   Results: No synthetic rescue of temperature-sensitivity was observed. Instead we found an additive phenotype, indicating gene products function in separate biological processes. These findings are in agreement with a recent genomic screen which found that RTS1 deletion in several ts-replication factor strains led to increased temperature-sensitivity. Conclusions: We find no evidence that Rts1 is involved in the dephosphorylation of DNA replication initiation factors.

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