scholarly journals SAF-A promotes origin licensing and replication fork progression to ensure robust DNA replication

2021 ◽  
Author(s):  
Caitlin Connolly ◽  
Saori Takahashi ◽  
Hisashi Miura ◽  
Ichiro Hiratani ◽  
Nick Gilbert ◽  
...  
Keyword(s):  
Dna Replication ◽  
Biological Activities ◽  
Timing Analysis ◽  
Replication Timing ◽  
Synthesis Rate ◽  
Open Chromatin ◽  
Replication Fork Progression ◽  
Chromosome Domains ◽  
Origin Licensing ◽  
Attachment Factor

The organisation of chromatin is closely intertwined with biological activities of chromosome domains, including transcription and DNA replication status. Scaffold attachment factor A (SAF-A), also known as Heteronuclear Ribonucleoprotein Protein U (HNRNPU), contributes to the formation of open chromatin structure. Here we demonstrate that SAF-A promotes the normal progression of DNA replication, and enables resumption of replication after inhibition. We report that cells depleted for SAF-A show reduced origin licensing in G1 phase, and consequently reduced origin activation frequency in S phase. Replication forks also progress less consistently in cells depleted for SAF-A, contributing to reduced DNA synthesis rate. Single-cell replication timing analysis revealed two distinct effects of SAF-A depletion: first, the boundaries between early- and late-replicating domains become more blurred; and second, SAF-A depletion causes replication timing changes that tend to bring regions of discordant domain compartmentalisation and replication timing into concordance. Associated with these defects, SAF-A-depleted cells show elevated -H2AX formation and tend to enter quiescence. Overall we find that SAF-A protein promotes robust DNA replication to ensure continuing cell proliferation.

SAF-A promotes origin licensing and replication fork progression to ensure robust DNA replication

2021 ◽  
Author(s):  
Caitlin Connolly ◽  
Saori Takahashi ◽  
Hisashi Miura ◽  
Ichiro Hiratani ◽  
Nick Gilbert ◽  
...  
Keyword(s):  
Dna Replication ◽  
Biological Activities ◽  
Timing Analysis ◽  
Replication Timing ◽  
Synthesis Rate ◽  
Open Chromatin ◽  
Replication Fork Progression ◽  
Chromosome Domains ◽  
Origin Licensing ◽  
In Cells

The organisation of chromatin is closely intertwined with biological activities of chromosome domains, including transcription and DNA replication status. Scaffold attachment factor A (SAF-A), also known as Heteronuclear Ribonucleoprotein Protein U (HNRNPU), contributes to the formation of open chromatin structure. Here we demonstrate that SAF-A promotes the normal progression of DNA replication, and enables resumption of replication after inhibition. We report that cells depleted for SAF-A show reduced origin licensing in G1 phase, and consequently reduced origin activation frequency in S phase. Replication forks progress slowly in cells depleted for SAF-A, also contributing to reduced DNA synthesis rate. Single-cell replication timing analysis revealed that the boundaries between early- and late- replicating domains are blurred in cells depleted for SAF-A. Associated with these defects, SAF-A-depleted cells show elevated gH2A phosphorylation and tend to enter quiescence. Overall we find that SAF-A protein promotes robust DNA replication to ensure continuing cell proliferation.

scholarly journals Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila

Genetics ◽  
2021 ◽  
Author(s):  
Souradip Das ◽  
Madison Caballero ◽  
Tatyana Kolesnikova ◽  
Igor Zhimulev ◽  
Amnon Koren ◽  
...  
Keyword(s):  
Dna Replication ◽  
Copy Number ◽  
Genome Stability ◽  
Timing Analysis ◽  
Replication Timing ◽  
Critical Function ◽  
Replication Fork Progression ◽  
Polyploid Cells ◽  
Genomic Regions ◽  
Insight Into

Abstract Regulation of DNA replication and copy number is necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rap1-interacting factor 1 (Rif1) is a key regulator of RT and has a critical function in copy number control in polyploid cells. Previously, we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 acts to promote late replication, which is necessary for SUUR-dependent underreplication. Our work provides new insight into the process of UR and its links to RT.

scholarly journals Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila.

2021 ◽  
Author(s):  
Souradip Das ◽  
Madison Caballero ◽  
Tatyana Kolesnikova ◽  
Igor Zhimulev ◽  
Amnon Koren ◽  
...  
Keyword(s):  
Dna Replication ◽  
Copy Number ◽  
Genome Stability ◽  
Timing Analysis ◽  
Replication Timing ◽  
Critical Function ◽  
Replication Fork Progression ◽  
Polyploid Cells ◽  
Genomic Regions ◽  
Insight Into

Regulation of DNA replication and copy number are necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rif1 is key regulator of RT and has a critical function in copy number control in polyploid cells. In a previous study (Munden et al., 2018), we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 functions both upstream and downstream of SUUR to promote UR. Our work provides new mechanistic insight into the process of UR and its links to RT.

scholarly journals Efficient, quick and easy-to-use DNA replication timing analysis with START-R suite

2020 ◽  
Vol 2 (2) ◽  
Author(s):  
Djihad Hadjadj ◽  
Thomas Denecker ◽  
Eva Guérin ◽  
Su-Jung Kim ◽  
Fabien Fauchereau ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cell Fate ◽  
Web Application ◽  
Timing Analysis ◽  
Mutant Cell ◽  
Replication Timing ◽  
Experimental Conditions ◽  
Replication Process ◽  
Time Required ◽  
Dna Replication Timing

Abstract DNA replication must be faithful and follow a well-defined spatiotemporal program closely linked to transcriptional activity, epigenomic marks, intranuclear structures, mutation rate and cell fate determination. Among the readouts of the spatiotemporal program of DNA replication, replication timing analyses require not only complex and time-consuming experimental procedures, but also skills in bioinformatics. We developed a dedicated Shiny interactive web application, the START-R (Simple Tool for the Analysis of the Replication Timing based on R) suite, which analyzes DNA replication timing in a given organism with high-throughput data. It reduces the time required for generating and analyzing simultaneously data from several samples. It automatically detects different types of timing regions and identifies significant differences between two experimental conditions in ∼15 min. In conclusion, START-R suite allows quick, efficient and easier analyses of DNA replication timing for all organisms. This novel approach can be used by every biologist. It is now simpler to use this method in order to understand, for example, whether ‘a favorite gene or protein’ has an impact on replication process or, indirectly, on genomic organization (as Hi-C experiments), by comparing the replication timing profiles between wild-type and mutant cell lines.

scholarly journals New, easy, quick and efficient DNA replication timing analysis by high-throughput approaches

2019 ◽  
Author(s):  
Djihad Hadjadj ◽  
Thomas Denecker ◽  
Eva Guérin ◽  
Su-Jung Kim ◽  
Fabien Fauchereau ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cell Fate ◽  
Web Application ◽  
Global Analysis ◽  
Timing Analysis ◽  
Replication Timing ◽  
Experimental Conditions ◽  
Spatio Temporal ◽  
Dna Replication Timing ◽  
Temporal Program

AbstractDNA replication must be faithful and follow a well-defined spatio-temporal program closely linked to transcriptional activity, epigenomic marks, intra-nuclear structures, mutation rate and cell fate determination. Among the readouts of the DNA replication spatio-temporal program, replication timing (RT) analyses require complex, precise and time-consuming experimental procedures, and the study of large-size computer files. We improved the RT protocol to speed it up and increase its quality and reproducibility. Also, we partly automated the RT protocol and developed a user-friendly software: the START-R suite (Simple Tool for the Analysis of the Replication Timing based on R). START-R suite is an open source web application using an R script and an HTML interface to analyze DNA replication timing in a given cell line with microarray or deep-sequencing results. This novel approach can be used by every biologist without requiring specific knowledge in bioinformatics. It also reduces the time required for generating and analyzing simultaneously data from several samples. START-R suite detects constant timing regions (CTR) but also, and this is a novelty, it identifies temporal transition regions (TTR) and detects significant differences between two experimental conditions. The informatic global analysis requires less than 10 minutes.

scholarly journals Replication timing alterations in leukemia affect clinically relevant chromosome domains

2019 ◽  
Vol 3 (21) ◽  
pp. 3201-3213 ◽  
Author(s):  
Juan Carlos Rivera-Mulia ◽  
Takayo Sasaki ◽  
Claudia Trevilla-Garcia ◽  
Naoto Nakamichi ◽  
David J. H. F. Knapp ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Dna Replication ◽  
Clinical Outcome ◽  
B Cell ◽  
Replication Timing ◽  
B Cell Differentiation ◽  
Chromosome Domains ◽  
Key Points ◽  
Dna Replication Timing ◽  
Genome Alteration

Key Points DNA replication timing of >100 pediatric leukemic samples identified BCP-ALL subtype-specific genome alteration signatures. Comparative analyses identified features of specific stages of B-cell differentiation and potential associations with clinical outcome.

scholarly journals The Genetic Architecture of DNA Replication Timing in Human Pluripotent Stem Cells

2020 ◽  
Author(s):  
Qiliang Ding ◽  
Matthew M. Edwards ◽  
Michelle L. Hulke ◽  
Alexa N. Bracci ◽  
Ya Hu ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Sequences ◽  
Timing Analysis ◽  
Replication Timing ◽  
Histone Code ◽  
Replication Initiation ◽  
Histone Hyperacetylation ◽  
Stem Cell Lines ◽  
Early Replication ◽  
Dna Replication Timing

AbstractDNA replication follows a strict spatiotemporal program that intersects with chromatin structure and gene regulation. However, the genetic basis of the mammalian DNA replication timing program is poorly understood1–3. To systematically identify genetic regulators of DNA replication timing, we exploited inter-individual variation in 457 human pluripotent stem cell lines from 349 individuals. We show that the human genome’s replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs4) – base-pair-resolution sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, to affect replication timing. Analysis of rtQTL locations reveals a histone code for replication initiation, composed of bivalent histone H3 trimethylation marks on a background of histone hyperacetylation. The H3 trimethylation marks are individually repressive yet synergize to promote early replication. We further identify novel positive and negative regulators of DNA replication timing, the former comprised of pluripotency-related transcription factors while the latter involve boundary elements. Human replication timing is controlled by a multi-layered mechanism that operates on target DNA sequences, is composed of dozens of effectors working combinatorially, and follows principles analogous to transcription regulation: a histone code, activators and repressors, and a promoter-enhancer logic.

scholarly journals Loss of histone H3.3 results in DNA replication defects and altered origin dynamics in C. elegans

2019 ◽  
Author(s):  
Maude Strobino ◽  
Joanna M. Wenda ◽  
Florian A. Steiner
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Replication Fork ◽  
Replication Timing ◽  
Embryonic Cell ◽  
Stress Conditions ◽  
Replication Origins ◽  
C Elegans ◽  
Replication Fork Progression ◽  
Histone H3.3

AbstractHistone H3.3 is a replication-independent variant of histone H3 with important roles in development, differentiation and fertility. Here we show that loss of H3.3 results in replication defects in Caenorhabditis elegans embryos. To characterize these defects, we adapt methods to determine replication timing, map replication origins, and examine replication fork progression. Our analysis of the spatiotemporal regulation of DNA replication shows that despite the very rapid embryonic cell cycle, the genome is replicated from early and late firing origins and is partitioned into domains of early and late replication. We find that under temperature stress conditions, additional replication origins become activated. Moreover, loss of H3.3 results in impaired replication fork progression around origins, which is particularly evident at stress-activated origins. These replication defects are accompanied by replication checkpoint activation, a prolonged cell cycle, and increased lethality in checkpoint-compromised embryos. Our comprehensive analysis of DNA replication in C. elegans reveals the genomic location of replication origins and the dynamics of their firing, and uncovers a role of H3.3 in the regulation of replication origins under stress conditions.

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