scholarly journals Mitotic CDK promotes replisome disassembly, fork breakage, and complex DNA rearrangements

2018 ◽  
Author(s):  
Lin Deng ◽  
R. Alex. Wu ◽  
Olga V. Kochenova ◽  
David Pellman ◽  
Johannes C. Walter
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Chromosomal Rearrangements ◽  
Cyclin B1 ◽  
Template Switching ◽  
List Type ◽  
End Joining ◽  
Dna Rearrangements ◽  
Egg Extracts ◽  
The Impact

SUMMARYDNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. Although the events that trigger these errors are not well understood, one candidate is mitotic entry before the completion of DNA replication. To address the impact of mitosis on DNA replication, we employed Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive these extracts into mitosis, the E3 ubiquitin ligase TRAIP promotes ubiquitylation of the replicative CMG (CDC45/MCM2–7/GINS) helicase at stalled forks and at forks that have completed DNA synthesis. In both cases, ubiquitylation is followed by CMG extraction from chromatin by the CDC48/p97 ATPase. At stalled forks, CMG removal results in fork breakage and complex end joining events involving deletions and template-switching. Our results identify TRAIP-dependent replisome disassembly as a novel trigger of replication fork collapse and propose it underlies complex DNA rearrangements in mitosis.HIGHLIGHTSTRAIP-dependent MCM7 ubiquitylation removes all CMGs from chromatin in mitosisCMG unloading from stalled forks causes replication fork breakageReplication fork breakage in mitosis causes complex rearrangementsNew model of replication fork collapse

scholarly journals Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

2020 ◽  
Vol 6 (38) ◽  
pp. eabc0330 ◽  
Author(s):  
D. T. Gruszka ◽  
S. Xie ◽  
H. Kimura ◽  
H. Yardimci
Keyword(s):  
Dna Replication ◽  
Real Time ◽  
Single Molecule ◽  
Replication Fork ◽  
Epigenetic Inheritance ◽  
Replication Forks ◽  
Replication Fork Progression ◽  
Egg Extracts ◽  
Fork Stalling ◽  
The Moment

During replication, nucleosomes are disrupted ahead of the replication fork, followed by their reassembly on daughter strands from the pool of recycled parental and new histones. However, because no previous studies have managed to capture the moment that replication forks encounter nucleosomes, the mechanism of recycling has remained unclear. Here, through real-time single-molecule visualization of replication fork progression in Xenopus egg extracts, we determine explicitly the outcome of fork collisions with nucleosomes. Most of the parental histones are evicted from the DNA, with histone recycling, nucleosome sliding, and replication fork stalling also occurring but at lower frequencies. Critically, we find that local histone recycling becomes dominant upon depletion of endogenous histones from extracts, revealing that free histone concentration is a key modulator of parental histone dynamics at the replication fork. The mechanistic details revealed by these studies have major implications for our understanding of epigenetic inheritance.

scholarly journals Replication Fork Uncoupling Causes Nascent Strand Degradation and Fork Reversal

2021 ◽  
Author(s):  
Tamar Kavlashvili ◽  
James M Dewar
Keyword(s):  
Dna Replication ◽  
Replication Fork ◽  
Genome Stability ◽  
Replicative Helicase ◽  
Before And After ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Biochemical Approach ◽  
Key Steps ◽  
Xenopus Egg Extracts

Genotoxins cause nascent strand degradation (NSD) and fork reversal during DNA replication. NSD and fork reversal are crucial for genome stability and exploited by chemotherapeutic approaches. However, it is unclear how NSD and fork reversal are triggered. Additionally, the fate of the replicative helicase during these processes is unknown. We developed a biochemical approach to study synchronous, localized NSD and fork reversal using Xenopus egg extracts. We show that replication fork uncoupling stimulates NSD of both nascent strands and progressive conversion of uncoupled forks to reversed forks. The replicative helicase remains bound during NSD and fork reversal, indicating that both processes take place behind the helicase. Unexpectedly, NSD occurs before and after fork reversal, indicating that multiple degradation steps take place. Overall, our data show that uncoupling causes NSD and fork reversal and identify key steps involved in these processes.

scholarly journals Transcription drives DNA replication initiation and termination in human cells

2018 ◽  
Author(s):  
Yu-Hung Chen ◽  
Sarah Keegan ◽  
Malik Kahli ◽  
Peter Tonzi ◽  
David Fenyö ◽  
...  
Keyword(s):  
Dna Replication ◽  
Rna Polymerase Ii ◽  
Replication Fork ◽  
Human Cells ◽  
Replication Initiation ◽  
Origin Firing ◽  
Dna Replication Initiation ◽  
Origin Activation ◽  
Dna Replication Origins ◽  
The Impact

ABSTRACTThe locations of active DNA replication origins in the human genome, and the determinants of origin activation, remain controversial. Additionally, neither the predominant sites of replication termination nor the impact of transcription on replication-fork mobility have been defined. We demonstrate that replication initiation occurs preferentially in the immediate vicinity of the transcription start site of genes occupied by high levels of RNA polymerase II, ensuring co-directional replication of the most highly transcribed genes. Further, we demonstrate that dormant replication origin firing represents the global activation of pre-existing origins. We also show that DNA replication naturally terminates at the polyadenylation site of transcribed genes. During replication stress, termination is redistributed to gene bodies, generating a global reorientation of replication relative to transcription. Our analysis provides a unified model for the coupling of transcription with replication initiation and termination in human cells.

scholarly journals The non-homologous end joining factor Ku orchestrates replication fork resection and fine-tunes Rad51-mediated fork restart

2017 ◽  
Author(s):  
Ana Teixeira-Silva ◽  
Anissia Ait Saada ◽  
Ismail Iraqui ◽  
Marina Charlotte Nocente ◽  
Karine Fréon ◽  
...  
Keyword(s):  
Replication Fork ◽  
Double Strand Breaks ◽  
Fine Tuning ◽  
List Type ◽  
End Joining ◽  
Strand Breaks ◽  
Replication Restart ◽  
Dna End Resection ◽  
Non Homologous End Joining ◽  
End Resection

AbstractReplication requires Homologous Recombination (HR) to stabilize and restart terminally-arrested forks. HR-mediated fork processing requires single stranded DNA (ssDNA) gaps and not necessarily Double Strand Breaks. We used genetic and molecular assays to investigate fork-resection and restart at dysfunctional, unbroken forks in Schizosaccharomyces pombe. We found that fork-resection is a two-step process coordinated by the non-homologous end joining factor Ku. An initial resection mediated by MRN/Ctp1 removes Ku from terminally-arrested forks, generating ~ 110 bp sized gaps obligatory for subsequent Exo1-mediated long-range resection and replication restart. The lack of Ku results in slower fork restart, excessive resection, and impaired RPA recruitment. We propose that terminally-arrested forks undergo fork reversal, providing a single DNA end for Ku binding which primes RPA-coated ssDNA. We uncover an unprecedented role for Ku in orchestrating resection of unbroken forks and in fine-tuning HR-mediated replication restart.Ku orchestrates a two-steps DNA end-resection of terminally-arrested and unbroken forksMRN/Ctp1 removes Ku from terminally-arrested forks to initiate fork-resectiona ~110 bp sized ssDNA gap is sufficient and necessary to promote fork restart.The lack of Ku decreases ssDNA RPA-coating, and slows down replication fork restart.

scholarly journals Mechanism of replication-coupled DNA-protein crosslink proteolysis by SPRTN and the proteasome

2018 ◽  
Author(s):  
Alan Gao ◽  
Nicolai B. Larsen ◽  
Justin L. Sparks ◽  
Irene Gallina ◽  
Matthias Mann ◽  
...  
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Dna Lesions ◽  
List Type ◽  
Genomic Integrity ◽  
Dna Metabolism ◽  
Single Stranded Dna ◽  
Egg Extracts ◽  
Protein Crosslinks ◽  
Bulky Dna Lesions

SummaryDNA-protein crosslinks (DPCs) are bulky DNA lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S-phase removal of DPCs, but how SPRTN activity is coupled to DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is triggered by single-stranded DNA, a byproduct of DNA replication. In contrast, SPRTN-mediated DPC degradation is independent of DPC polyubiquitylation but requires polymerase extension of a nascent strand to the lesion. Thus, SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms and together promote replication across immovable protein barriers.HighlightsThe proteasome, in addition to SPRTN, degrades DPCs during DNA replicationProteasome-dependent DPC degradation requires DPC ubiquitylationDPC ubiquitylation is triggered by ssDNA and does not require the replisomeSPRTN-dependent DPC degradation is a post-replicative process

scholarly journals Ubiquitin/SUMO modification of PCNA promotes replication fork progression in Xenopus laevis egg extracts

2005 ◽  
Vol 171 (6) ◽  
pp. 947-954 ◽  
Author(s):  
Craig A. Leach ◽  
W. Matthew Michael
Keyword(s):  
Xenopus Laevis ◽  
Replication Fork ◽  
Nuclear Antigen ◽  
Molecular Gas ◽  
Chromosomal Replication ◽  
Dna Templates ◽  
Replication Fork Progression ◽  
Egg Extracts ◽  
The Impact ◽  
Proliferating Cell

The homotrimeric DNA replication protein proliferating cell nuclear antigen (PCNA) is regulated by both ubiquitylation and sumoylation. We study the appearance and the impact of these modifications on chromosomal replication in frog egg extracts. Xenopus laevis PCNA is modified on lysine 164 by sumoylation, monoubiquitylation, and diubiquitylation. Sumoylation and monoubiquitylation occur during the replication of undamaged DNA, whereas diubiquitylation occurs specifically in response to DNA damage. When lysine 164 modification is prevented, replication fork movement through undamaged DNA slows down and DNA polymerase δ fails to associate with replicating chromatin. When sumoylation alone is prevented, replication occurs normally and neither monoubiquitylation nor sumoylation are required for the replication of simple single-strand DNA templates. Our findings expand the repertoire of functions for PCNA ubiquitylation and sumoylation by elucidating a role for these modifications during the replication of undamaged DNA. Furthermore, they suggest that PCNA monoubiquitylation serves as a molecular gas pedal that controls the speed of replisome movement during S phase.

TARGETING CYCLIN B1 WITH ANTISENSE OLIGONUCLETOTIDES- COATED SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES

2018 ◽  
Vol 6 (10) ◽  
Author(s):  
Hosam Zaghloul ◽  
Doaa A. Shahin ◽  
Ibrahim El- Dosoky ◽  
Mahmoud E. El-awady ◽  
Fardous F. El-Senduny ◽  
...  
Keyword(s):  
Iron Oxide ◽  
Iron Oxide Nanoparticles ◽  
Cellular Uptake ◽  
Superparamagnetic Iron Oxide ◽  
Cyclin B1 ◽  
Superparamagnetic Iron Oxide Nanoparticles ◽  
Oxide Nanoparticles ◽  
Mcf7 Cells ◽  
M Phase ◽  
The Impact

Antisense oligonucleotides (ASO) represent an attractive trend as specific targeting molecules but sustain poor cellular uptake meanwhile superparamagnetic iron oxide nanoparticles (SPIONs) offer stability of ASO and improved cellular uptake. In the present work we aimed to functionalize SPIONs with ASO targeting the mRNA of Cyclin B1 which represents a potential cancer target and to explore its anticancer activity. For that purpose, four different SPIONs-ASO conjugates, S-M (1–4), were designated depending on the sequence of ASO and constructed by crosslinking carboxylated SPIONs to amino labeled ASO. The impact of S-M (1–4) on the level of Cyclin B1, cell cycle, ROS and viability of the cells were assessed by flowcytometry. The results showed that S-M3 and S-M4 reduced the level of Cyclin B1 by 35 and 36%, respectively. As a consequence to downregulation of Cyclin B1, MCF7 cells were shown to be arrested at G2/M phase (60.7%). S-M (1–4) led to the induction of ROS formation in comparison to the untreated control cells. Furthermore, S-M (1–4) resulted in an increase in dead cells compared to the untreated cells and SPIONs-treated cells. In conclusion, targeting Cyclin B1 with ASO-coated SPIONs may represent a specific biocompatible anticancer strategy.

scholarly journals Histone H3 Lysine 4 Methylation Marks Postreplicative Human Cytomegalovirus Chromatin

2012 ◽  
Vol 86 (18) ◽  
pp. 9817-9827 ◽  
Author(s):  
Alexandra Nitzsche ◽  
Charlotte Steinhäußer ◽  
Katrin Mücke ◽  
Christina Paulus ◽  
Michael Nevels
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Histone Modification ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Histone H3 ◽  
Viral Dna ◽  
The Impact ◽  
Preferential Incorporation

In the nuclei of permissive cells, human cytomegalovirus genomes form nucleosomal structures initially resembling heterochromatin but gradually switching to a euchromatin-like state. This switch is characterized by a decrease in histone H3 K9 methylation and a marked increase in H3 tail acetylation and H3 K4 methylation across the viral genome. We used ganciclovir and a mutant virus encoding a reversibly destabilized DNA polymerase to examine the impact of DNA replication on histone modification dynamics at the viral chromatin. The changes in H3 tail acetylation and H3 K9 methylation proceeded in a DNA replication-independent fashion. In contrast, the increase in H3 K4 methylation proved to depend widely on viral DNA synthesis. Consistently, labeling of nascent DNA using “click chemistry” revealed preferential incorporation of methylated H3 K4 into viral (but not cellular) chromatin during or following DNA replication. This study demonstrates largely selective epigenetic tagging of postreplicative human cytomegalovirus chromatin.

scholarly journals Using Satellite Data for CBRN (Chemical, Biological, Radiological, and Nuclear) Threat Detection, Monitoring, and Modelling

2021 ◽  
Author(s):  
Gary Sutlieff ◽  
Lucy Berthoud ◽  
Mark Stinchcombe
Keyword(s):  
Satellite Data ◽  
Meteorological Data ◽  
Atmospheric Composition ◽  
Chemical Detection ◽  
List Type ◽  
Data Types ◽  
Wide Range ◽  
Future Technologies ◽  
The Impact ◽  
Near Future

Abstract CBRN (Chemical, Biological, Radiological, and Nuclear) threats are becoming more prevalent, as more entities gain access to modern weapons and industrial technologies and chemicals. This has produced a need for improvements to modelling, detection, and monitoring of these events. While there are currently no dedicated satellites for CBRN purposes, there are a wide range of possibilities for satellite data to contribute to this field, from atmospheric composition and chemical detection to cloud cover, land mapping, and surface property measurements. This study looks at currently available satellite data, including meteorological data such as wind and cloud profiles, surface properties like temperature and humidity, chemical detection, and sounding. Results of this survey revealed several gaps in the available data, particularly concerning biological and radiological detection. The results also suggest that publicly available satellite data largely does not meet the requirements of spatial resolution, coverage, and latency that CBRN detection requires, outside of providing terrain use and building height data for constructing models. Lastly, the study evaluates upcoming instruments, platforms, and satellite technologies to gauge the impact these developments will have in the near future. Improvements in spatial and temporal resolution as well as latency are already becoming possible, and new instruments will fill in the gaps in detection by imaging a wider range of chemicals and other agents and by collecting new data types. This study shows that with developments coming within the next decade, satellites should begin to provide valuable augmentations to CBRN event detection and monitoring. Article Highlights There is a wide range of existing satellite data in fields that are of interest to CBRN detection and monitoring. The data is mostly of insufficient quality (resolution or latency) for the demanding requirements of CBRN modelling for incident control. Future technologies and platforms will improve resolution and latency, making satellite data more viable in the CBRN management field

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